US20030175861A1

US20030175861A1 - Monoterpene synthases from grand fir (abies grandis)

Info

Publication number: US20030175861A1
Application number: US10/025,145
Authority: US
Inventors: C.L. Steele; Joerg Bohlmann; Rodney Croteau; Michael Phillips
Original assignee: Washington State University Research Foundation
Current assignee: Washington State University Research Foundation
Priority date: 1997-07-11
Filing date: 2001-12-19
Publication date: 2003-09-18
Also published as: WO2001007565A2; US6429014B1; WO2001007565A3

Abstract

cDNAs encoding gymnosperm monoterpene synthases have been isolated and sequenced, and the corresponding amino acid sequences have been determined. Modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding a monoterpene synthase of the invention. Thus, systems and methods are provided for the recombinant expression of recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts.

Description

RELATED APPLICATIONS

The present application is a continuation-in-part of international application serial number U.S. Ser. No. 98/14528, filed on Jul. 10, 1998, which claims benefit of priority from U.S. provisional application serial No. 60/052,249 filed on Jul. 11, 1997. This invention was funded in part by grant GM-3135A from the National Institutes of Health and by grant 97-35302-4432 from the United States Department of Agriculture. The government has certain rights in this invention.[0001]

FIELD OF THE INVENTION

The present invention relates to nucleic acid sequences which code for monoterpene synthases from gymnosperm plant species, in particular from Grand fir ( Abies grandis), including (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, limonene synthase, myrcene synthase, and pinene synthase, to vectors containing the sequences, to host cells containing the sequences, to plant seeds expressing the sequences and to methods of producing recombinant monoterpene synthases and their mutants.

BACKGROUND OF THE INVENTION

Chemical defense of conifer trees against bark beetles and their associated fungal pathogens relies primarily upon constitutive and inducible oleoresin biosynthesis (Johnson, M. A., and Croteau, R. (1987) in Ecology and Metabolism of Plant Lipids (Fuller, G., and Nes, W. D., eds.) pp. 76-91, American Chemical Society Symposium Series 325, Washington, D.C.; Gijzen, M., Lewinsohn, E., Savage, T. J., and Croteau, R. B. (1993) in Bioactive Volatile Compounds from Plants(Teranishi, R., Buttery, R. G., and Sugisawa, H., eds.) pp. 8-22, American Chemical Society Symposium Series 525, Washington, D.C.). This defensive secretion is a complex mixture of monoterpene and sesquiterpene olefins (turpentine) and diterpene resin acids (rosin) that is synthesized constitutively in the epithelial cells of specialized structures, such as resin ducts and blisters or, in the case of induced oleoresin formation, in undifferentiated cells surrounding wound sites (Lewinsohn, E., Gijzen, M., Savage, T. J., and Croteau, R. (1991) Plant Physiol. 96:38-43). The volatile fraction of conifer oleoresin, which is toxic to both bark beetles and their fungal associates (Raffa, K. F., Berryman, A. A., Simasko, J., Teal, W., and Wong, B. L. (1985) Environ. Entomol. 14:552-556), may consist of up to 30 different monoterpenes (Lewinsohn, E., Savage, T. J., Gijzen, M., and Croteau, R. (1993) Phytochem. Anal. 4:220-225), including acyclic types (e.g., myrcene), monocyclic types (e.g., limonene) and bicyclic types (e.g., pinenes) (FIG. 1). Although the oleoresin is toxic, many bark beetle species nevertheless employ turpentine volatiles in host selection and can convert various monoterpene components into aggregation or sex pheromones to promote coordinated mass attack of the host (Gijzen, M., Lewinsohn, E., Savage, T. J., and Croteau, R. B. (1993) in Bioactive Volatile Compounds from Plants (Teranishi, R., Buttery, R. G., and Sugisawa, H., eds.) pp. 8-22, American Chemical Society Symposium Series 525, Washington, D.C.; Byers, J. A. (1995) in Chemical Ecology of Insects 2 (Cardé, R. T., and Bell, W. J., eds.) pp. 154-213, Chapman and Hall, New York). In Grand fir (Abies grandis), increased formation of oleoresin monoterpenes, sesquiterpenes and diterpenes is induced by bark beetle attack (Lewinsohn, E., Gijzen, M., Savage, T. J., and Croteau, †R. (1991) Plant Physiol. 96:38-43; Raffa, K. F., and Berrynan, A. A. (1982) Can. Entomol. 114:797-810; Lewinsohn, E., Gijzen, M., and Croteau, R. (1991) Plant Physiol. 96:44-49), and this inducible defense response is mimicked by mechanically wounding sapling stems (Lewinsohn, E., Gijzen, M., Savage, T. J., and Croteau, R. (1991) Plant Physiol. 96:38-43; Lewinsohn, E., Gijzen, M., and Croteau, R. (1991) Plant Physiol. 96:44-49; Funk, C., Lewinsohn, E., Stofer Vogel, B., Steele C., and Croteau, R. (1994) Plant Physiol. 106:999-1005). Therefore, Grand fir has been developed as a model system to study the biochemical and molecular genetic regulation of constitutive and inducible terpene biosynthesis in conifers (Steele, C., Lewinsohn, E., and Croteau, R. (1995) Proc. Natl. Acad. Sci. USA 92:4164-4168).

Most monoterpenes are derived from geranyl diphosphate, the ubiquitous C ₁₀intermediate of the isoprenoid pathway, by synthases which catalyze the divalent metal ion-dependent ionization (to 1, FIG. 1) and isomerization of this substrate to enzyme-bound linalyl diphosphate which, following rotation about C2-C3, undergoes a second ionization (to 2, FIG. 1) followed by cyclization to the α-terpinyl cation, the first cyclic intermediate en route to both monocyclic and bicyclic products (Croteau, R., and Cane, D. E. (1985) Methods Enzymol. 110:383-405; Croteau, R. (1987) Chem. Rev. 87:929-954) (FIG. 1). Acyclic monoterpenes, such as myrcene, may arise by deprotonation of

carbocations

1 or 2, whereas the isomerization step to linalyl diphosphate is required in the case of cyclic types, such as limonene and pinenes, which cannot be derived from geranyl diphosphate directly because of the geometric impediment of the trans-double bond at C2-C3 (Croteau, R., and Cane, D. E. (1985) Methods Enzymol. 110:383-405; Croteau, R. (1987) Chem. Rev. 87:929-954). Many monoterpene synthases catalyze the formation of multiple products, including acyclic, monocyclic and bicyclic types, by variations on this basic mechanism (Gambliel, H., and Croteau, R. (1984) J. Biol. Chem. 259:740-748; Croteau, R., Satterwhite, D. M., Cane, D. E., and Chang, C. C. (1988) J. Biol. Chem. 263:10063-10071; Croteau, R., and Satterwhite, D. M. (1989) J. Biol. Chem. 264:15309-15315). For example, (−)-limonene synthase, the principal monoterpene synthase of spearmint (Mentha spicata) and peppermint (M. x piperita), produces small amounts of myrcene, (−)-α-pinene and (−)-β-pinene in addition to the monocyclic product Rajaonarivony, J. I. M., Gershenzon, J., and Croteau, R. (1992) Arch. Biochem. Biophys. 296:49-57; Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024. Conversely, six different inducible monoterpene synthase activities have been demonstrated in extracts of wounded Grand fir stem (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289:267-273) indicating that formation of acyclic, monocyclic and bicyclic monoterpenes in this species involves several genes encoding distinct catalysts. The inducible (−)-pinene synthase has been purified (Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173), and isotopically sensitive branching experiments employed to demonstrate that this enzyme synthesizes both (−)-α- and (−)-β-pinene (Wagschal, K., Savage, T. J., and Croteau, R. (1991) Tetrahedron 47:5933-5944).

Deciphering the molecular genetic control of oleoresinosis and examining structure-function relationships among the monoterpene synthases of Grand fir requires isolation of the cDNA species encoding these key enzymes. Although a protein-based cloning strategy was recently employed to acquire a cDNA for the major wound-inducible diterpene synthase from Grand fir, abietadiene synthase (Funk, C., Lewinsohn, E., Stofer Vogel, B., Steele C., and Croteau, R. (1994) Plant Physiol. 106:999-1005; LaFever, R. E., Stofer Vogel, B., and Croteau, R. (1994) Arch. Biochem. Biophys. 313:139-149; Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268), all attempts at the reverse genetic approach to cloning of Grand fir monoterpene synthases have failed (Steele, C., Lewinsohn, E., and Croteau, R. (1995) Proc. Natl. Acad. Sci. USA 92:4164-4168). As an alternative, a similarity-based PCR strategy was developed (Steele, C., Lewinsohn, E., and Croteau, R. (1995) Proc. Natl. Acad. Sci. USA 92:4164-4168) that employed sequence information from terpene synthases of angiosperm origin, namely a monoterpene synthase, (−)-4S-limonene synthase, from spearmint (Mentha spicata, Lamiaceae) (Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024), a sesquiterpene synthase, 5-epi-aristolochene synthase, from tobacco (Nicotiana tabacum, Solanaceae) (Facchini, P. J., and Chappell, J. (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092), and a diterpene synthase, casbene synthase, from castor bean (Ricinus communis, Euphorbiaceae) (Mau, C. J. D., and West, C. A. (1994) Proc. Natl. Acad. Sci. USA 91:8497-8501).

Monoterpenes have significant potential for cancer prevention and treatment. Monoterpenes such as limonene, perillyl alcohol, carvone, geraniol and farnesol not only reduce tumor incidence and slow tumor proliferation, but have also been reported to cause regression of established solid tumors by initiating apoptosis (Mills J. J., Chari R. S., Boyer I. J., Gould M. N., Jirtle R. L., Cancer Res., 55:979-983, 1995). Terpenes have activity against cancers such as mammary, colon, and prostate. Clinical trials are being pursued (Seachrist L, J. NIH Res. 8:43) in patients with various types of advanced cancers to validate the health benefits of dietary terpenes for humans. However, terpenes are present in Western diets at levels that are probably inadequate for any significant preventive health benefits. Daily supplementation of the diet with a terpene concentrate (10-20 g/day) would appear to be the most rational strategy for dietary therapy of diagnosed cases of cancer. This invention envisages the production of such nutritionally beneficial terpenes in, for example, vegetable oils consumed daily via the engineering of relevant genes from Grand fir into oil seed crop plants such as oil seed brassica (canola), soybean and corn.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: [0007]
FIG. 1 is a schematic representation depicting the mechanism for the conversion of geranyl diphosphate to myrcene, (−)-limonene, β-phellandrene, (−)-α-pinene and (−)-β-pinene by monoterpene synthases from Grand fir. Formation of the monocyclic and bicyclic products requires preliminary isomerization of geranyl diphosphate to linalyl diphosphate. The acyclic product could be formed from either geranyl diphosphate or linalyl diphosphate via [0008] carbocations 1 or 2. OPP denotes the diphosphate moiety.
FIG. 2 is a sequence comparison of plant terpene synthases. A three-letter designation (Tps) for the gene family is proposed with sub-groups (Tpsa through Tpsf) defined by a minimum of 40% amino acid identity between members. [0009]
FIG. 3 depicts a GLC-MS analysis of the products of the recombinant protein encoded by AG2.2 (SEQ ID NO: 1), the sequence of the protein encoded by clone AG2.2 being set forth in SEQ ID NO: 2. The GLC profile of the total pentane-soluble products generated from geranyl diphosphate when incubated with a cell-free extract of [0010] E. coli XL1-Blue/pGAG2.2 is illustrated (FIG. 3A), as are the mass fragmentation patterns for the monoterpene product with R_t=12.22 min (FIG. 3B) and for authentic myrcene (FIG. 3C).
FIG. 4 depicts a GLC-MS analysis of the products of the recombinant protein encoded by AG3.18 (SEQ ID NO: 3), the sequence of the protein encoded by clone AG3.18 (SEQ ID NO: 3) being set forth in SEQ ID NO: 4. The GLC profile of the total pentane-soluble products generated from geranyl diphosphate when incubated with a cell-free extract of [0011] E. coli XL1-Blue/pGAG3.18 is illustrated (FIG. 4A), as are the mass fragmentation patterns (selected ion mode) for the monoterpene products with R_t=11.34 min (FIG. 4B), and R_t=13.37 min (FIG. 4D), and for authentic α-pinene (FIG. 4C) and authentic β-pinene (FIG. 4E).
FIG. 5 depicts a GLC-MS analysis of the products of the recombinant protein encoded by AG10 (SEQ ID NO: 5), the sequence of the protein encoded by clone AG10 (SEQ ID NO: 5) being set forth in SEQ ID NO: 6. The GLC profile of the total pentane-soluble products generated from geranyl diphosphate when incubated with a cell-free extract of [0012] E. coli BL21(DE3)/pSBAG10 is illustrated (FIG. 5A), as are the mass fragmentation patterns for the principal monoterpene product with R_t=13.93 min (FIG. 5B) and for authentic limonene (FIG. 5C).
FIG. 6A depicts a total ion chromatogram of monoterpene products derived from geranyl diphosphate by a (−)-camphene synthase of the invention. [0013]
FIG. 6B depicts the mass spectrum and retention time for the principal enzyme product shown in FIG. 6A. [0014]
FIG. 6C depicts the mass spectrum and retention time for the authentic camphene standard. [0015]
FIG. 7A depicts a total ion chromatogram of monoterpene products derived from geranyl diphosphate by a (−)-β-phellandrene synthase of the invention. [0016]
FIG. 7B depicts the mass spectrum and retention time for the principal enzyme product shown in FIG. 7A. [0017]
FIG. 7C depicts the mass spectrum and retention time for the authentic β-phellandrene standard. [0018]
FIG. 8A depicts a total ion chromatogram of monoterpene products derived from geranyl diphosphate by a terpinolene synthase of the invention. [0019]
FIG. 8B depicts the mass spectrum and retention time for the principal enzyme product shown in FIG. 8A. [0020]
FIG. 8C depicts the mass spectrum and retention time for the authentic terpinolene standard. [0021]
FIG. 9A depicts a total ion chromatogram of monoterpene products derived from geranyl diphosphate by a (−)-limonene/(−)-α-pinene synthase of the invention. [0022]
FIG. 9B depicts the mass spectrum and retention time for the principal enzyme product shown in FIG. 9A. [0023]
FIG. 9C depicts the mass spectrum and retention time for the authentic α-pinene standard.[0024]

SUMMARY OF THE INVENTION

In accordance with the foregoing, cDNAs encoding (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, myrcene synthase, (−)-limonene synthase and (−)-pinene synthase from Grand fir ([0025] Abies grandis) have been isolated and sequenced, and the corresponding amino acid sequences have been deduced. Accordingly, the present invention relates to isolated DNA sequences which code for the expression of monoterpene synthases, including (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, myrcene synthase, (−)-limonene synthase and (−)-pinene synthase, and to isolated nucleic acid molecules that hybridize to portions of Grand fir monoterpene synthase cDNAs, as described more fully herein. In another aspect, the present invention relates to isolated monoterpene synthases, including isolated (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase and (−)-limonene/(−)-α-pinene synthase. In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence, e.g., a DNA sequence which codes for a monoterpene synthase such as (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, myrcene synthase, (−)-limonene synthase or (−)-pinene synthase, or for a base sequence sufficiently complementary to at least a portion of DNA or RNA encoding a monoterpene synthase such as (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, myrcene synthase, (−)-limonene synthase or (−)-pinene synthase to enable hybridization therewith (e.g., antisense RNA or fragments of DNA complementary to a portion of DNA or RNA molecules encoding (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, myrcene synthase, (−)-limonene synthase or (−)-pinene synthase which are useful as polymerase chain reaction primers or as probes for any of the foregoing synthases or related genes). In yet other aspects of the invention, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention. Thus, the present invention provides for the recombinant expression of (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, myrcene synthase, (−)-limonene synthase and (−)-pinene synthase, and the inventive concepts may be used to facilitate the production, isolation and purification of significant quantities of recombinant (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, myrcene synthase, (−)-limonene synthase and (−)-pinene synthase (or of their primary enzyme products) for subsequent use, to obtain expression or enhanced expression of (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, myrcene synthase, (−)-limonene synthase and (−)-pinene synthase in microorganisms, animals or plants (including, but not limited to, Brassica, cotton, soybean, safflower, sunflower, coconut, palm, wheat, barley, rice, corn, oats, amaranth, pumpkin, squash, sesame, poppy, grape, mung beans, peanut, peas, beans, broad beans, chick peas, lentils, radish, alfalfa, cocoa, coffee, tree nuts, spinach, culinary herbs, berries, stone fruit and citrus), or may be otherwise employed in an environment where the regulation or expression of the foregoing monoterpene synthases is desired for the production of these synthases, or their enzyme products, or derivatives thereof. In another aspect, the present invention relates to manipulation of monoterpene production to enhance resistance to insects and/or accumulate nutritionally beneficial monoterpenes in oil seeds (such as seeds from Brassica, cotton, soybean, safflower, sunflower, coconut, palm, wheat, barley, rice, corn, oats, amaranth, pumpkin, squash, sesame, poppy, grape, mung beans, peanut, peas, beans, broad beans, chick peas, lentils, radish, alfalfa, cocoa, coffee and tree nuts) and other food stuffs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As used herein, the terms “amino acid” and “amino acids” refer to all naturally occurring L-α-amino acids or their residues. The amino acids are identified by either the single-letter or three-letter designations:



Asp	D	aspartic acid	Ile	I	isoleucine
Thr	T	threonine	Leu	L	leucine
Ser	S	serine	Tyr	Y	tyrosine
Glu	E	glutamic acid	Phe	F	phenylalanine
Pro	P	proline	His	H	histidine
Gly	G	glycine	Lys	K	lysine
Ala	A	alanine	Arg	R	arginine
Cys	C	cysteine	Trp	W	tryptophan
Val	V	valine	Gln	Q	glutamine
Met	M	methionine	Asn	N	asparagine

As used herein, the term “nucleotide” means a monomeric unit of DNA or RNA containing a sugar moiety (pentose), a phosphate and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1′ carbon of pentose) and that combination of base and sugar is called a nucleoside. The base characterizes the nucleotide with the four bases of DNA being adenine (“A”), guanine (“G”), cytosine (“C”) and thymine (“T”). Inosine (“I”) is a synthetic base that can be used to substitute for any of the four, naturally-occurring bases (A, C, G or T). The four RNA bases are A,G,C and uracil (“U”). The nucleotide sequences described herein comprise a linear array of nucleotides connected by phosphodiester bonds between the 3′ and 5′ carbons of adjacent pentoses. [0027]
The term “percent identity” means the percentage of amino acids or nucleotides that occupy the same relative position when two amino acid sequences, or two nucleic acid sequences are aligned side by side. [0028]
The term “percent similarity” is a statistical measure of the degree of relatedness of two compared protein sequences. The percent similarity is calculated by a computer program that assigns a numerical value to each compared pair of amino acids based on chemical similarity (e.g., whether the compared amino acids are acidic, basic, hydrophobic, aromatic, etc.) and/or evolutionary distance as measured by the minimum number of base pair changes that would be required to convert a codon encoding one member of a pair of compared amino acids to a codon encoding the other member of the pair. Calculations are made after a best fit alignment of the two sequences have been made empirically by iterative comparison of all possible alignments. (Henikoff, S. and Henikoff, J. G., [0029] Proc. Nat'l. Acad. Sci. USA 89:10915-10919, 1992).
“Oligonucleotide” refers to short length single or double stranded sequences of deoxyribonucleotides linked via phosphodiester bonds. The oligonucleotides are chemically synthesized by known methods and purified, for example, on polyacrylamide gels. [0030]
The term “myrcene synthase” is used herein to mean an enzyme capable of generating multiple monoterpenes from geranyl diphosphate. The principal and characteristic monoterpene synthesized by myrcene synthase is myrcene, which constitutes at least about 50% of the monoterpene mixture synthesized by myrcene synthase from geranyl diphosphate. [0031]
The term “(−)-limonene synthase” is used herein to mean an enzyme capable of generating multiple monoterpenes from geranyl diphosphate. The principal and characteristic monoterpene synthesized by (−)-limonene synthase is (−)-limonene, which constitutes at least about 60% of the monoterpene mixture synthesized by (−)-limonene synthase from geranyl diphosphate. [0032]
The term “(−)-pinene synthase” is used herein to mean an enzyme capable of generating multiple monoterpenes from geranyl diphosphate. The principal and characteristic monoterpene synthesized by (−)-pinene synthase is (−)-pinene, which comprises at least about 50% of the monoterpene mixture synthesized by (−)-pinene synthase from geranyl diphosphate. [0033]
The term “(−)-camphene synthase” is used herein to mean an enzyme capable of generating multiple monoterpenes from geranyl diphosphate. The principal and characteristic monoterpene synthesized by (−)-camphene synthase is (−)-camphene, which comprises at least about 50% of the monoterpene mixture synthesized by (−)-camphene synthase from geranyl diphosphate. [0034]
The term “(−)-β-phellandrene synthase” is used herein to mean an enzyme capable of generating multiple monoterpenes from geranyl diphosphate. The principal and characteristic monoterpene synthesized by (−)-β-phellandrene synthase is (−)-β-phellandrene, which comprises at least about 50% of the monoterpene mixture synthesized by (−)-β-phellandrene synthase from geranyl diphosphate. [0035]
The term “terpinolene synthase” is used herein to mean an enzyme capable of generating multiple monoterpenes from geranyl diphosphate. The principal and characteristic monoterpene synthesized by terpinolene synthase is terpinolene which comprises at least about 40% of the monoterpene mixture synthesized by terpinolene synthase from geranyl diphosphate. [0036]
The term (−)-limonene/(−)-α-pinene synthase is used herein to mean an enzyme capable of generating multiple monoterpenes from geranyl diphosphate. The principal and characteristic monoterpenes synthesized by (−)-limonene/(−)-α-pinene synthase are (−)-limonene and (−)-α-pinene which comprise at least about 35% and 25%, respectively, of the monoterpene mixture synthesized by (−)-limonene/(−)-α-pinene synthase from geranyl diphosphate. [0037]
The abbreviation “SSPE” refers to a buffer used in nucleic acid hybridization solutions. The 20× (twenty times concentrate) stock SSPE buffer solution is prepared as follows: dissolve 175.3 grams of NaCl, 27.6 grams of NaH[0038] ₂PO₄H₂O and 7.4 grams of EDTA in 800 millilitres of H₂O. Adjust the pH to pH 7.4 with NaOH. Adjust the volume to one liter with H₂O.
The abbreviation “SSC” refers to a buffer used in nucleic acid hybridization solutions. One liter of the 20× (twenty times concentrate) stock SSC buffer solution (pH 7.0) contains 175.3 g sodium chloride and 88.2 g sodium citrate. [0039]
The terms “alteration”, “amino acid sequence alteration”, “variant” and “amino acid sequence variant” refer to monoterpene synthase molecules with some differences in their amino acid sequences as compared to the corresponding, native, i.e., naturally-occurring, monoterpene synthases. Ordinarily, the variants will possess at least about 70% homology with the corresponding native monoterpene synthases, and preferably, they will be at least about 80% homologous with the corresponding, native monoterpene synthases. The amino acid sequence variants of the monoterpene synthases falling within this invention possess substitutions, deletions, and/or insertions at certain positions. Sequence variants of monoterpene synthases may be used to attain desired enhanced or reduced enzymatic activity, modified regiochemistry or stereochemistry, or altered substrate utilization or product distribution. [0040]
Substitutional monoterpene synthase variants are those that have at least one amino acid residue in the native monoterpene synthase sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. Substantial changes in the activity of the monoterpene synthase molecules of the present invention may be obtained by substituting an amino acid with a side chain that is significantly different in charge and/or structure from that of the native amino acid. This type of substitution would be expected to affect the structure of the polypeptide backbone and/or the charge or hydrophobicity of the molecule in the area of the substitution. [0041]
Moderate changes in the activity of the monoterpene synthase molecules of the present invention would be expected by substituting an amino acid with a side chain that is similar in charge and/or structure to that of the native molecule. This type of substitution, referred to as a conservative substitution, would not be expected to substantially alter either the structure of the polypeptide backbone or the charge or hydrophobicity of the molecule in the area of the substitution. [0042]
Insertional monoterpene synthase variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in the native monoterpene synthase molecule. Immediately adjacent to an amino acid means connected to either the α-carboxy or α-amino functional group of the amino acid. The insertion may be one or more amino acids. Ordinarily, the insertion will consist of one or two conservative amino acids. Amino acids similar in charge and/or structure to the amino acids adjacent to the site of insertion are defined as conservative. Alternatively, this invention includes insertion of an amino acid with a charge and/or structure that is substantially different from the amino acids adjacent to the site of insertion. [0043]
Deletional variants are those where one or more amino acids in the native monoterpene synthase molecules have been removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the monoterpene synthase molecule. [0044]
The terms “biological activity”, “biologically active”, “activity” and “active” refer to the ability of the monoterpene synthases of the present invention to convert geranyl diphosphate to a group of monoterpenes, of which myrcene is the principal and characteristic monoterpene synthesized by myrcene synthase, (−)-limonene is the principal and characteristic monoterpene synthesized by (−)-limonene synthase, (−)-pinene is the principal and characteristic monoterpene synthesized by (−)-pinene synthase, (−)-camphene is the principal and characteristic monoterpene synthesized by (−)-camphene synthase, (−)-β-phellandrene is the principal and characteristic monoterpene synthesized by (−)-β-phellandrene synthase, terpinolene is the principal and characteristic monoterpene synthesized by terpinolene synthase, and (−)-limonene and (−)-α-pinene are the principal and characteristic monoterpenes synthesized by (−)-limonene/(−)-α-pinene synthase. The monoterpenes produced by the monoterpene synthases of the present invention are as measured in an enzyme activity assay, such as the assay described in Example 3. Amino acid sequence variants of the monoterpene synthases of the present invention may have desirable altered biological activity including, for example, altered reaction kinetics, substrate utilization product distribution or other characteristics such as regiochemistry and stereochemistry. [0045]
The terms “DNA sequence encoding”, “DNA encoding” and “nucleic acid encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the translated polypeptide chain. The DNA sequence thus codes for the amino acid sequence. [0046]
The terms “replicable expression vector” and “expression vector” refer to a piece of DNA, usually double-stranded, which may have inserted into it a piece of foreign DNA. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of or coincidental with the host chromosomal DNA, and several copies of the vector and its inserted (foreign) DNA may be generated. In addition, the vector contains the necessary elements that permit translating the foreign DNA into a polypeptide. Many molecules of the polypeptide encoded by the foreign DNA can thus be rapidly synthesized. [0047]
The terms “transformed host cell,” “transformed” and “transformation” refer to the introduction of DNA into a cell. The cell is termed a “host cell”, and it may be a prokaryotic or a eukaryotic cell. Typical prokaryotic host cells include various strains of [0048] E coli. Typical eukaryotic host cells are plant cells, such as maize cells, yeast cells, insect cells or animal cells. The introduced DNA is usually in the form of a vector containing an inserted piece of DNA. The introduced DNA sequence may be from the same species as the host cell or from a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign DNA and some DNA derived from the host species.
In accordance with the present invention, cDNAs encoding myrcene synthase (SEQ ID NO: 1), (−)-pinene synthase (SEQ ID NO: 3) and (−)-limonene synthase (SEQ ID NO: 5) from Grand fir ([0049] Abies grandis) were isolated and sequenced in the following manner. Based on comparison of sequences of limonene synthase from spearmint (Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024), 5-epi-aristolochene synthase from tobacco (Facchini, P. J., and Chappell, J. (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092), and casbene synthase from castor bean (Mau, C. J. D., and West, C. A. (1994) Proc. Natl. Acad. Sci. USA 91:8497-8501), four conserved regions were identified for which a set of consensus, degenerate primers were synthesized: Primer A (SEQ ID NO: 7), Primer B (SEQ ID NO: 8), Primer C (SEQ ID NO: 9) and Primer D (SEQ ID NO: 10). Primers A (SEQ ID NO: 7), B (SEQ ID NO: 8), and D (SEQ ID NO: 10) were sense primers, while Primer C (SEQ ID NO: 9), was an antisense primer. Each of the sense primers, A (SEQ ID NO: 7), B (SEQ ID NO: 8) and D (SEQ ID NO: 10), was used for PCR in combination with antisense primer C (SEQ ID NO: 9) by employing a broad range of amplification conditions. Analysis of the PCR reaction products by agarose gel electrophoresis revealed that only the combination of primers C (SEQ ID NO: 9) and D (SEQ ID NO: 10) generated a specific PCR product of approximately 110 bps.
The 110 bps PCR product was gel purified, ligated into a plasmid, and transformed into [0050] E. coli XL1-Blue cells. Plasmid DNA was prepared from 41 individual transformants and the inserts were sequenced. Four different insert sequences were identified, and were designated as probes 1 (SEQ ID NO: 11), 2 (SEQ ID NO: 12), 4 (SEQ ID NO: 13) and 5 (SEQ ID NO: 14). Probes 1 (SEQ ID NO: 11), 2 (SEQ ID NO: 12), 4 (SEQ ID NO: 13) and 5 (SEQ ID NO: 14) were used to screen a cDNA library made from mRNA extracted from wounded Grand fir stems, and the longest clone that hybridized to each of these probes was isolated and sequenced. Thus, clone AG1.28 (SEQ ID NO: 15) is the longest cDNA clone that hybridized to probe 1 (SEQ ID NO: 11), clone AG2.2 (SEQ ID NO: 1) is the longest cDNA clone that hybridized to probe 2 (SEQ ID NO: 12), clone AG4.30 (SEQ ID NO: 17) is the longest cDNA clone that hybridized to probe 4 (SEQ ID NO: 13), and clone AG5.9 (SEQ ID NO: 19) is the longest cDNA clone that hybridized to probe 5 (SEQ ID NO: 14).
Truncated clone AG1.28 (SEQ ID NO: 15) resembled most closely in size and sequence (72% similarity, 49% identity) a diterpene cyclase, abietadiene synthase, from Grand fir. Clones AG4.30 (SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19) encode sesquiterpene synthases. Sequence and functional analysis of clone AG2.2 (SEQ ID NO: 1) revealed that it encoded the monoterpene synthase, myrcene synthase. [0051]
Alignment of the four new terpene synthase cDNA sequences AG1.28 (SEQ ID NO: 15), AG2.2 (SEQ ID NO: 1), AG4.30 (SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19) with that for abietadiene synthase (Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996) [0052] J. Biol. Chem. 271:23262-23268) allowed the identification of several, conserved sequence motifs. Two new sense PCR primers, primer E (SEQ ID NO: 21) and primer F (SEQ ID NO: 22) were designed based on the sequence of the conserved protein sequence motifs. A new antisense PCR primer, primer G (SEQ ID NO: 23), was designed based on limited sequence information available from pinene synthase. The combination of primer E (SEQ ID NO: 21) and primer G (SEQ ID NO: 23) amplified a cDNA product of 1022 bps, which was designated as probe 3 (SEQ ID NO: 24).
Probe 3 (SEQ ID NO: 24) was used to screen a cDNA library made from mRNA extracted from wounded Grand fir stems. Hybridization of 10[0053] ⁵Grand fir λZAP II cDNA clones with probe 3 (SEQ ID NO: 24) yielded two types of signals comprised of about 400 strongly positive clones and an equal number of weak positives, indicating that the probe recognized more than one type of cDNA. Thirty-four of the former clones and eighteen of the latter were purified, the inserts were selected by size (2.0-2.5 kb), and the in vivo excised clones were partially sequenced from both ends. Those clones which afforded weak hybridization signals were shown to contain inserts that were either identical to myrcene synthase clone AG2.2 (SEQ ID NO: 1) or exhibited no significant sequence similarity to terpene synthases.
Clones which gave strong hybridization signals segregated into distinct sequence groups represented by clone AG3.18 (SEQ ID NO: 3) and clone AG10 (SEQ ID NO: 5). Both AG3.18 (SEQ ID NO: 3) and AG10 (SEQ ID NO: 5) were subcloned into plasmid expression vectors and expressed in [0054] E. coli. When extracts of the induced cells were tested for terpene synthase activity with all of the potential prenyl diphosphate substrates, only geranyl diphosphate was utilized. Extracts from E. coli containing the AG10 (SEQ ID NO: 5) expression construct converted geranyl diphosphate to the (−)-4S enantiomer of limonene as the major product, indicating that AG10 (SEQ ID NO: 5) encodes (−)-limonene synthase. Similar analysis of the monoterpene products generated from geranyl diphosphate by cell-free extracts of E. coli containing the AG3.18 (SEQ ID NO: 3) insert ligated into an expression vector revealed the presence of a 42:58% mixture of α-pinene and β-pinene, the same product ratio previously described for the purified, native (−)-pinene synthase from Grand fir. These data indicate that AG3.18 (SEQ ID NO: 3) encodes (−)-pinene synthase.
Additionally, cDNA molecules encoding (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase were isolated and characterized as described in Example 11. [0055]
The isolation of cDNAs encoding (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase and myrcene synthase permits the development of efficient expression systems for these functional enzymes; provides useful tools for examining the developmental regulation of monoterpene biosynthesis; permits investigation of the reaction mechanism(s) of these unusual, multiproduct enzymes, and permits the isolation of other monoterpene synthases including (−)-camphene synthases, (−)-β-phellandrene synthases, terpinolene synthases, (−)-limonene/(−)-α-pinene synthases, (−)-limonene synthases, (−)-pinene synthases and myrcene synthases. [0056]
The isolation of the (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase and myrcene synthase cDNAs also permits the transformation of a wide range of organisms in order to introduce monoterpene biosynthesis de novo, or to modify endogenous monoterpene biosynthesis. [0057]
Substitution of the presumptive targeting sequence of the cloned monoterpene synthases (e.g., SEQ ID NO: 2, [0058] amino acids 1 to 61; SEQ ID NO: 4, amino acids 1 to 61; SEQ ID NO: 6, amino acids 1 to 66) with other transport sequences well known in the art (see, e.g., von Heijne et al., Eur. J. Biochem. 180:535-545, 1989; Stryer, Biochemistry, W. H. Freeman and Company, New York, N.Y., p. 769 [1988]) may be employed to direct the cloned monoterpene synthases of the invention to other cellular or extracellular locations.
In addition to the native monoterpene synthase amino acid sequences, sequence variants produced by deletions, substitutions, mutations and/or insertions are intended to be within the scope of the invention except insofar as limited by the prior art. The monoterpene synthase amino acid sequence variants of this invention may be constructed by mutating the DNA sequences that encode the wild-type synthases, such as by using techniques commonly referred to as site-directed mutagenesis. Nucleic acid molecules encoding the monoterpene synthases of the present invention can be mutated by a variety of PCR techniques well known to one of ordinary skill in the art. See, e.g., “PCR Strategies”, M. A. Innis, D. H. Gelfand and J. J. Sninsky, eds., 1995, Academic Press, San Diego, Calif. (Chapter 14); “PCR Protocols: A Guide to Methods and Applications”, M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White, eds., Academic Press, NY (1990). [0059]
By way of non-limiting example, the two primer system utilized in the Transformer Site-Directed Mutagenesis kit from Clontech, may be employed for introducing site-directed mutants into the monoterpene synthase genes of the present invention. Following denaturation of the target plasmid in this system, two primers are simultaneously annealed to the plasmid; one of these primers contains the desired site-directed mutation, the other contains a mutation at another point in the plasmid resulting in elimination of a restriction site. Second strand synthesis is then carried out, tightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of [0060] E. coli. Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (thereby linearizing the unmutated plasmids), and then retransformed into E. coli. This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids. The tight linkage of the two mutations and the subsequent linearization of unmutated plasmids results in high mutation efficiency and allows minimal screening. Following synthesis of the initial restriction site primer, this method requires the use of only one new primer type per mutation site. Rather than prepare each positional mutant separately, a set of “designed degenerate” oligonucleotide primers can be synthesized in order to introduce all of the desired mutations at a given site simultaneously. Transformants can be screened by sequencing the plasmid DNA through the mutagenized region to identify and sort mutant clones. Each mutant DNA can then be restricted and analyzed by electrophoresis on Mutation Detection Enhancement gel (J. T. Baker) to confirm that no other alterations in the sequence have occurred (by band shift comparison to the unmutagenized control).
The verified mutant duplexes in the pET (or other) overexpression vector can be employed to transform [0061] E. coli such as strain E. coli BL21(DE3)pLysS, for high level production of the mutant protein, and purification by standard protocols. The method of FAB-MS mapping can be employed to rapidly check the fidelity of mutant expression. This technique provides for sequencing segments throughout the whole protein and provides the necessary confidence in the sequence assignment. In a mapping experiment of this type, protein is digested with a protease (the choice will depend on the specific region to be modified since this segment is of prime interest and the remaining map should be identical to the map of unmutagenized protein). The set of cleavage fragments is fractionated by microbore BPLC (reversed phase or ion exchange, again depending on the specific region to be modified) to provide several peptides in each fraction, and the molecular weights of the peptides are determined by FAB-MS. The masses are then compared to the molecular weights of peptides expected from the digestion of the predicted sequence, and the correctness of the sequence quickly ascertained. Since this mutagenesis approach to protein modification is directed, sequencing of the altered peptide should not be necessary if the MS agrees with prediction. If necessary to verify a changed residue, CAD-tandem MS/MS can be employed to sequence the peptides of the mixture in question, or the target peptide purified for subtractive Edman degradation or carboxypeptidase Y digestion depending on the location of the modification.
In the design of a particular site directed mutagenesis, it is generally desirable to first make a non-conservative substitution (e.g., Ala for Cys, His or Glu) and determine if activity is greatly impaired as a consequence. The properties of the mutagenized protein are then examined with particular attention to the kinetic parameters of K[0062] _mand k_catas sensitive indicators of altered function, from which changes in binding and/or catalysis per se may be deduced by comparison to the native enzyme. If the residue is by this means demonstrated to be important by activity impairment, or knockout, then conservative substitutions can be made, such as Asp for Glu to alter side chain length, Ser for Cys, or Arg for His. For hydrophobic segments, it is largely size that is usefully altered, although aromatics can also be substituted for alkyl side chains. Changes in the normal product distribution can indicate which step(s) of the reaction sequence have been altered by the mutation. Modification of the hydrophobic pocket can be employed to change binding conformations for substrates and result in altered regiochemistry and/or stereochemistry.
Other site directed mutagenesis techniques may also be employed with the nucleotide sequences of the invention. For example, restriction endonuclease digestion of DNA followed by ligation may be used to generate deletion variants of (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase and myrcene synthase, as described in section 15.3 of Sambrook et al. ([0063] Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York, N.Y. [1989]). A similar strategy may be used to construct insertion variants, as described in section 15.3 of Sambrook et al., supra.
Oligonucleotide-directed mutagenesis may also be employed for preparing substitution variants of this invention. It may also be used to conveniently prepare the deletion and insertion variants of this invention. This technique is well known in the art as described by Adelman et al. ([0064] DNA 2:183 [1983]); Sambrook et al., supra; “Current Protocols in Molecular Biology”, 1991, Wiley (NY), F. T. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. D. Seidman, J. A. Smith and K. Struhl, eds.
Generally, oligonucleotides of at least 25 nucleotides in length are used to insert, delete or substitute two or more nucleotides in the nucleic acid molecules encoding monoterpene synthases of the invention. An optimal oligonucleotide will have 12 to 15 perfectly matched nucleotides on either side of the nucleotides coding for the mutation. To mutagenize nucleic acids encoding wild-type monoterpene synthases of the invention, the oligonucleotide is annealed to the single-stranded DNA template molecule under suitable hybridization conditions. A DNA polymerizing enzyme, usually the Klenow fragment of [0065] E. coli DNA polymerase 1, is then added. This enzyme uses the oligonucleotide as a primer to complete the synthesis of the mutation-bearing strand of DNA. Thus, a heteroduplex molecule is formed such that one strand of DNA encodes the wild-type synthase inserted in the vector, and the second strand of DNA encodes the mutated form of the synthase inserted into the same vector. This heteroduplex molecule is then transformed into a suitable host cell.
Mutants with more than one amino acid substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If however, the amino acids are located some distance from each other (separated by more than ten amino acids, for example) it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions. An alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: wild-type monoterpene synthase DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on. [0066]
A gene encoding (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase or myrcene synthase may be incorporated into any organism (intact plant, animal, microbe, etc.), or cell culture derived therefrom, that produces geranyl diphosphate. A (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase or myrcene synthase gene may be introduced into any organism for a variety of purposes including, but not limited to: production of (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase or myrcene synthase, or their products; production or modification of flavor and aroma properties; improvement of defense capability, and the alteration of other ecological interactions mediated by (−)-camphene, (−)-β-phellandrene, terpinolene, myrcene, (−)-limonene, (−)-pinene, or their derivatives. [0067]
Eukaryotic expression systems may be utilized for the production of (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase and myrcene synthase since they are capable of carrying out any required posttranslational modifications and of directing the enzymes to the proper membrane location. A representative eukaryotic expression system for this purpose uses the recombinant baculovirus, [0068] Autographa californica nuclear polyhedrosis virus (AcNPV; M. D. Summers and G. E. Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures [1986]; Luckow et al., Bio-technology 6:47-55 [1987]) for expression of the terpenoid synthases of the invention. Infection of insect cells (such as cells of the species Spodoptera frugiperda) with the recombinant baculoviruses allows for the production of large amounts of the monoterpene synthase proteins. In addition, the baculovirus system has other important advantages for the production of recombinant monoterpene synthases. For example, baculoviruses do not infect humans and can therefore be safely handled in large quantities. In the baculovirus system, a DNA construct is prepared including a DNA segment encoding (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase or myrcene synthase and a vector. The vector may comprise the polyhedron gene promoter region of a baculovirus, the baculovirus flanking sequences necessary for proper cross-over during recombination (the flanking sequences comprise about 200-300 base pairs adjacent to the promoter sequence) and a bacterial origin of replication which permits the construct to replicate in bacteria. The vector is constructed so that (i) the DNA segment is placed adjacent (or operably linked or “downstream” or “under the control of”) to the polyhedron gene promoter and (ii) the promoter/monoterpene synthase combination is flanked on both sides by 200-300 base pairs of baculovirus DNA (the flanking sequences).
To produce the monoterpene synthase DNA construct, a cDNA clone encoding the full length (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase or myrcene synthase is obtained using methods such as those described herein. The DNA construct is contacted in a host cell with baculovirus DNA of an appropriate baculovirus (that is, of the same species of baculovirus as the promoter encoded in the construct) under conditions such that recombination is effected. The resulting recombinant baculoviruses encode the full (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase or myrcene synthase. For example, an insect host cell can be cotransfected or transfected separately with the DNA construct and a functional baculovirus. Resulting recombinant baculoviruses can then be isolated and used to infect cells to effect production of the monoterpene synthase. Host insect cells include, for example, [0069] Spodoptera frugiperda cells, that are capable of producing a baculovirus-expressed monoterpene synthase. Insect host cells infected with a recombinant baculovirus of the present invention are then cultured under conditions allowing expression of the baculovirus-encoded (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase or myrcene synthase. Monoterpene synthases thus produced are then extracted from the cells using methods known in the art.
Other eukaryotic microbes such as yeasts may also be used to practice this invention. The baker's yeast [0070] Saccharomyces cerevisiae, is a commonly used yeast, although several other strains are available. The plasmid YRp7 (Stinchcomb et al., Nature 282:39 [1979]; Kingsman et al., Gene 7:141 [1979]; Tschemper et al., Gene 10:157 [1980]) is commonly used as an expression vector in Saccharomyces. This plasmid contains the trp1 gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, such as strains ATCC No. 44,076 and PEP4-1 (Jones, Generics 85:12 [1977]). The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Yeast host cells are generally transformed using the polyethylene glycol method, as described by Hinnen (Proc. Natl. Acad. Sci. USA 75:1929 [1978]). Additional yeast transformation protocols are set forth in Gietz et al., N.A.R. 20(17) 1425(1992); Reeves et al., FEMS 99(2-3):193-197, (1992).
Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., [0071] J. Biol. Chem. 255:2073 [1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149 [1968]; Holland et al., Biochemistry 17:4900 [1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In the construction of suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3′ of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.
Cell cultures derived from multicellular organisms, such as plants, may be used as hosts to practice this invention. Transgenic plants can be obtained, for example, by transferring plasmids that encode (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase and/or myrcene synthase and a selectable marker gene, e.g., the kan gene encoding resistance to kanamycin, into [0072] Agrobacterium tumefaciens containing a helper Ti plasmid as described in Hoeckema et al., Nature 303:179-181 [1983] and culturing the Agrobacterium cells with leaf slices of the plant to be transformed as described by An et al., Plant Physiology 81:301-305 [1986]. Transformation of cultured plant host cells is normally accomplished through Agrobacterium tumefaciens, as described above. Cultures of mammalian host cells and other host cells that do not have rigid cell membrane barriers are usually transformed using the calcium phosphate method as originally described by Graham and Van der Eb (Virology 52:546 [1978]) and modified as described in sections 16.32-16.37 of Sambrook et al., supra. However, other methods for introducing DNA into cells such as Polybrene (Kawai and Nishizawa, Mol. Cell. Biol. 4:1172 [1984]), protoplast fusion (Schaffnier, Proc. Natl. Acad. Sci. USA 77:2163 [1980]), electroporation (Neumann et al., EMBO J. 1:841 [1982]), and direct microinjection into nuclei (Capecchi, Cell 22:479 [1980]) may also be used. Additionally, animal transformation strategies are reviewed in Monastersky G. M. and Robl, J. M., Strategies in Transgenic Animal Science, ASM Press, Washington, D.C., 1995. Transformed plant calli may be selected through the selectable marker by growing the cells on a medium containing, e.g., kanamycin, and appropriate amounts of phytohormone such as naphthalene acetic acid and benzyladenine for callus and shoot induction. The plant cells may then be regenerated and the resulting plants transferred to soil using techniques well known to those skilled in the art.
In addition, a gene regulating (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase or myrcene synthase production can be incorporated into the plant along with a necessary promoter which is inducible. In the practice of this embodiment of the invention, a promoter that only responds to a specific external or internal stimulus is fused to the target cDNA. Thus, the gene will not be transcribed except in response to the specific stimulus. As long as the gene is not being transcribed, its gene product is not produced. [0073]
An illustrative example of a responsive promoter system that can be used in the practice of this invention is the glutathione-S-transferase (GST) system in maize. GSTs are a family of enzymes that can detoxify a number of hydrophobic electrophilic compounds that often are used as pre-emergent herbicides (Weigand et al.; [0074] Plant Molecular Biology 7:235-243 [1986]). Studies have shown that the GSTs are directly involved in causing this enhanced herbicide tolerance. This action is primarily mediated through a specific 1.1 kb mRNA transcription product. In short, maize has a naturally occurring quiescent gene already present that can respond to external stimuli and that can be induced to produce a gene product. This gene has previously been identified and cloned. Thus, in one embodiment of this invention, the promoter is removed from the GST responsive gene and attached to a (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase or myrcene synthase gene that previously has had its native promoter removed. This engineered gene is the combination of a promoter that responds to an external chemical stimulus and a gene responsible for successful production of (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase or myrcene synthase.
In addition to the methods described above, several methods are known in the art for transferring cloned DNA into a wide variety of plant species, including gymnosperms, angiosperms, monocots and dicots (see, e.g., Glick and Thompson, eds., [0075] Methods in Plant Molecular Biology, CRC Press, Boca Raton, Fla. [1993]). Representative examples include electroporation-facilitated DNA uptake by protoplasts (Rhodes et al., Science 240(4849):204-207 [1988]); treatment of protoplasts with polyethylene glycol (Lyznik et al., Plant Molecular Biology 13:151-161 [1989]); and bombardment of cells with DNA laden microprojectiles (Klein et al., Plant Physiol. 91:440-444 [1989] and Boynton et al., Science 240(4858):1534-1538 [1988]). Additionally, plant transformation strategies and techniques are reviewed in Birch, R. G., Ann Rev Plant Phys Plant Mol Biol 48:297 (1997); Forester et al., Exp. Agric. 33:15-33 (1997). Minor variations make these technologies applicable to a broad range of plant species.
Each of these techniques has advantages and disadvantages. In each of the techniques, DNA from a plasmid is genetically engineered such that it contains not only the gene of interest, but also selectable and screenable marker genes. A selectable marker gene is used to select only those cells that have integrated copies of the plasmid (the construction is such that the gene of interest and the selectable and screenable genes are transferred as a unit). The screenable gene provides another check for the successful culturing of only those cells carrying the genes of interest. A commonly used selectable marker gene is neomycin phosphotransferase II (NPT II). This gene conveys resistance to kanamycin, a compound that can be added directly to the growth media on which the cells grow. Plant cells are normally susceptible to kanamycin and, as a result, die. The presence of the NPT II gene overcomes the effects of the kanamycin and each cell with this gene remains viable. Another selectable marker gene which can be employed in the practice of this invention is the gene which confers resistance to the herbicide glufosinate (Basta). A screenable gene commonly used is the β-glucokinase gene (GUS). The presence of this gene is characterized using a histochemical reaction in which a sample of putatively transformed cells is treated with a GUS assay solution. After an appropriate incubation, the cells containing the GUS gene turn blue. [0076]
The plasmid containing one or more of these genes is introduced into either plant protoplasts or callus cells by any of the previously mentioned techniques. If the marker gene is a selectable gene, only those cells that have incorporated the DNA package survive under selection with the appropriate phytotoxic agent. Once the appropriate cells are identified and propagated, plants are regenerated. Progeny from the transformed plants must be tested to insure that the DNA package has been successfully integrated into the plant genome. [0077]
Mammalian host cells may also be used in the practice of the invention. Examples of suitable mammalian cell lines include monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line 293S (Graham et al., [0078] J. Gen. Virol. 36:59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (Urlab and Chasin, Proc. Natl. Acad. Sci USA 77:4216 [1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod 23:243 [1980]); monkey kidney cells (CVI-76, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, BB 8065); mouse mammary tumor cells (MT 060562, ATCC CCL 51); rat hepatoma cells (HTC, MI.54, Baumann et al., J. Cell Biol. 85:1 [1980]); and TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44 [1982]). Expression vectors for these cells ordinarily include (if necessary) DNA sequences for an origin of replication, a promoter located in front of the gene to be expressed, a ribosome binding site, an RNA splice site, a polyadenylation site, and a transcription terminator site.
Promoters used in mammalian expression vectors are often of viral origin. These viral promoters are commonly derived from polyoma virus, [0079] Adenovirus 2, and most frequently Simian Virus 40 (SV40). The SV40 virus contains two promoters that are termed the early and late promoters. These promoters are particularly useful because they are both easily obtained from the virus as one DNA fragment that also contains the viral origin of replication (Fiers et al., Nature 273:113 [1978]). Smaller or larger SV40 DNA fragments may also be used, provided they contain the approximately 250-bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication.
Alternatively, promoters that are naturally associated with the foreign gene (homologous promoters) may be used provided that they are compatible with the host cell line selected for transformation. [0080]
An origin of replication may be obtained from an exogenous source, such as SV40 or other virus (e.g., Polyoma, Adeno, VSV, BPV) and inserted into the cloning vector. Alternatively, the origin of replication may be provided by the host cell chromosomal replication mechanism. If the vector containing the foreign gene is integrated into the host cell chromosome, the latter is often sufficient. [0081]
The use of a secondary DNA coding sequence can enhance production levels of (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase and myrcene synthase in transformed cell lines. The secondary coding sequence typically comprises the enzyme dihydrofolate reductase (DHFR). The wild-type form of DHFR is normally inhibited by the chemical methotrexate (MTX). The level of DHFR expression in a cell will vary depending on the amount of MTX added to the cultured host cells. An additional feature of DHFR that makes it particularly useful as a secondary sequence is that it can be used as a selection marker to identify transformed cells. Two forms of DHFR are available for use as secondary sequences, wild-type DHFR and MTX-resistant DHFR. The type of DHFR used in a particular host cell depends on whether the host cell is DHFR deficient (such that it either produces very low levels of DHFR endogenously, or it does not produce functional DHFR at all). DHFR-deficient cell lines such as the CHO cell line described by Urlaub and Chasin, supra, are transformed with wild-type DHFR coding sequences. After transformation, these DHFR-deficient cell lines express functional DHFR and are capable of growing in a culture medium lacking the nutrients hypoxanthine, glycine and thymidine. Nontransformed cells will not survive in this medium. [0082]
The MTX-resistant form of DHFR can be used as a means of selecting for transformed host cells in those host cells that endogenously produce normal amounts of functional DHFR that is MTX sensitive. The CHO-K1 cell line (ATCC No. CL 61) possesses these characteristics, and is thus a useful cell line for this purpose. The addition of MTX to the cell culture medium will permit only those cells transformed with the DNA encoding the MTX-resistant DHFR to grow. The nontransformed cells will be unable to survive in this medium. [0083]
Prokaryotes may also be used as host cells for the initial cloning steps of this invention. They are particularly useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated. Suitable prokaryotic host cells include [0084] E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325) E. coli X1776 (ATCC No. 31,537), and E. coli B; however many other strains of E. coli, such as HB101, JM101, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts. Prokaryotic host cells or other host cells with rigid cell walls are preferably transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation may be used for transformation of these cells. Prokaryote transformation techniques are set forth in Dower, W. J., in Genetic Engineering, Principles and Methods, 12:275-296, Plenum Publishing Corp., 1990; Hanahan et al., Meth. Enzymol., 204:63 (1991).
As a representative example, cDNA sequences encoding (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase or myrcene synthase may be transferred to the (His)[0085] ₆·Tag pET vector commercially available (from Novagen) for overexpression in E. coli as heterologous host. This pET expression plasmid has several advantages in high level heterologous expression systems. The desired cDNA insert is ligated in frame to plasmid vector sequences encoding six histidines followed by a highly specific protease recognition site (thrombin) that are joined to the amino terminus codon of the target protein. The histidine “block” of the expressed fusion protein promotes very tight binding to immobilized metal ions and permits rapid purification of the recombinant protein by immobilized metal ion affinity chromatography. The histidine leader sequence is then cleaved at the specific proteolysis site by treatment of the purified protein with thrombin, and the (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase and myrcene synthase again purified by immobilized metal ion affinity chromatography, this time using a shallower imidazole gradient to elute the recombinant synthases while leaving the histidine block still adsorbed. This overexpression-purification system has high capacity, excellent resolving power and is fast, and the chance of a contaminating E. coli protein exhibiting similar binding behavior (before and after thrombin proteolysis) is extremely small.
As will be apparent to those skilled in the art, any plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell may also be used in the practice of the invention. The vector usually has a replication site, marker genes that provide phenotypic selection in transformed cells, one or more promoters, and a polylinker region containing several restriction sites for insertion of foreign DNA. Plasmids typically used for transformation of [0086] E. coli include pBR322, pUC18, pUC19, pUCI18, pUC119, and Bluescript M13, all of which are described in sections 1.12-1.20 of Sambrook et al., supra. However, many other suitable vectors are available as well. These vectors contain genes coding for ampicillin and/or tetracycline resistance which enables cells transformed with these vectors to grow in the presence of these antibiotics.
The promoters most commonly used in prokaryotic vectors include the β-lactamase (penicillinase) and lactose promoter systems (Chang et al. [0087] Nature 375:615 [1978]; Itakura et al., Science 198:1056 [1977]; Goeddel et al., Nature 281:544 [1979]) and a tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 [1980]; EPO Appl. Publ. No. 36,776), and the alkaline phosphatase systems. While these are the most commonly used, other microbial promoters have been utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally into plasmid vectors (see Siebenlist et al., Cell 20:269 [1980]).
Many eukaryotic proteins normally secreted from the cell contain an endogenous secretion signal sequence as part of the amino acid sequence. Thus, proteins normally found in the cytoplasm can be targeted for secretion by linking a signal sequence to the protein. This is readily accomplished by ligating DNA encoding a signal sequence to the 5′ end of the DNA encoding the protein and then expressing this fusion protein in an appropriate host cell. The DNA encoding the signal sequence may be obtained as a restriction fragment from any gene encoding a protein with a signal sequence. Thus, prokaryotic, yeast, and eukaryotic signal sequences may be used herein, depending on the type of host cell utilized to practice the invention. The DNA and amino acid sequence encoding the signal sequence portion of several eukaryotic genes including, for example, human growth hormone, proinsulin, and proalbumin are known (see Stryer, [0088] Biochemistry W. H. Freeman and Company, New York, N.Y., p. 769 [1988]), and can be used as signal sequences in appropriate eukaryotic host cells. Yeast signal sequences, as for example acid phosphatase (Arima et al., Nuc. Acids Res. 11:1657 [1983]), α-factor, alkaline phosphatase and invertase may be used to direct secretion from yeast host cells. Prokaryotic signal sequences from genes encoding, for example, LamB or OmpF (Wong et al., Gene 68:193 [1988]), MalE, PhoA, or beta-lactamase, as well as other genes, may be used to target proteins from prokaryotic cells into the culture medium.
Trafficking sequences from plants, animals and microbes can be employed in the practice of the invention to direct the monoterpene synthase proteins of the present invention to the cytoplasm, endoplasmic reticulum, mitochondria or other cellular components, or to target the protein for export to the medium. These considerations apply to the overexpression of (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase and myrcene synthase, and to direction of expression within cells or intact organisms to permit gene product function in any desired location. [0089]
The construction of suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes and the monoterpene synthase DNA of interest are prepared using standard recombinant DNA procedures. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well known in the art (see, for example, Maniatis, supra, and Sambrook et al., supra). [0090]
As discussed above, (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, (−)-limonene synthase, (−)-pinene synthase and myrcene synthase variants are preferably produced by means of mutation(s) that are generated using the method of site-specific mutagenesis. This method requires the synthesis and use of specific oligonucleotides that encode both the sequence of the desired mutation and a sufficient number of adjacent nucleotides to allow the oligonucleotide to stably hybridize to the DNA template. [0091]
The foregoing may be more fully understood in connection with the following representative examples, in which “Plasmids” are designated by a lower case p followed by an alphanumeric designation. The starting plasmids used in this invention are either commercially available, publicly available on an unrestricted basis, or can be constructed from such available plasmids using published procedures. In addition, other equivalent plasmids are known in the art and will be apparent to the ordinary artisan. [0092]
“Digestion”, “cutting” or “cleaving” of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at particular locations in the DNA. These enzymes are called restriction endonucleases, and the site along the DNA sequence where each enzyme cleaves is called a restriction site. The restriction enzymes used in this invention are commercially available and are used according to the instructions supplied by the manufacturers. (See also sections 1.60-1.61 and sections 3.38-3.39 of Sambrook et al., supra.) [0093]
“Recovery” or “isolation” of a given fragment of DNA from a restriction digest means separation of the resulting DNA fragment on a polyacrylamide or an agarose gel by electrophoresis, identification of the fragment of interest by comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section containing the desired fragment, and separation of the gel from DNA. This procedure is known generally. For example, see Lawn et al. ([0094] Nucleic Acids Res. 9:6103-6114 [1982]), and Goeddel et al. (Nucleic Acids Res., supra).
The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention. [0095]

EXAMPLE 1

PCR-Based Generation of Probes for Cloning Certain Monoterpene Synthases From Grand fir ([0096] Abies grandis)
Substrates, Reagents and cDNA Library—[1-[0097] ³H]Geranyl diphosphate (250 Ci/mol) (Croteau, R., Alonso, W. R., Koepp, A. E., and Johnson, M. A. (1994) Arch. Biochem. Biophys. 309:184-192), [1-³H]farnesyl diphosphate (125 Ci/mol) (Dehal, S. S., and Croteau, R. (1988) Arch. Biochem. Biophys. 261:346-356) and [1-³H]geranylgeranyl diphosphate (120 Ci/mol) (LaFever, R. E., Stofer Vogel, B., and Croteau, R. (1994) Arch. Biochem. Biophys. 313:139-149) were prepared as described previously. Terpenoid standards were from our own collection. All other biochemicals and reagents were purchased from Sigma Chemical Co. or Aldrich Chemical Co., unless otherwise noted. Construction of the λZAP II cDNA library, using mRNA isolated from wounded Grand fir sapling stems, was described previously (Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268).
PCR-Based Probe Generation—Based on comparison of sequences of limonene synthase from spearmint (Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993) [0098] J. Biol. Chem. 268:23016-23024), 5-epi-aristolochene synthase from tobacco (Facchini, P. J., and Chappell, J. (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092), and casbene synthase from castor bean (Mau, C. J. D., and West, C. A. (1994) Proc. Natl. Acad. Sci. USA 91:8497-8501), four conserved regions were identified for which a set of consensus degenerate primers were synthesized: Primer A (SEQ ID NO: 7); Primer B (SEQ ID NO: 8); Primer C (SEQ ID NO: 9); Primer D (SEQ ID NO: 10). Primers A (SEQ ID NO: 7), B (SEQ ID NO: 8) and C (SEQ ID NO: 9) have been described previously (Steele, C., Lewinsohn, E. and Croteau, R., Proc. Nat'l. Acad. Sci. USA, 92: 4164-4168 (1995)); primer D (SEQ ID NO: 10) was designed based on the conserved amino acid sequence motif DD(T/I)(I/Y/F)D(A/V)Y(A/G)(SEQ ID NO: 25) of the above noted terpene synthases (Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024; Facchini, P. J., and Chappell, J. (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092; Mau, C. J. D., and West, C. A. (1994) Proc. Natl. Acad. Sci. USA 91:8497-8501).
Each of the sense primers, A (SEQ ID NO: 7), B (SEQ ID NO: 8) and D (SEQ ID NO: 10), was used for PCR in combination with antisense primer C (SEQ ID NO: 9) by employing a broad range of amplification conditions. PCR was performed in a total volume of 50 μl containing 20 mM Tris/HCl (tris(hydroxymethyl) aminomethane/HCl, pH 8.4), 50 mM KCl, 5 mM MgCl[0099] ₂, 200 μM of each dNTP, 1-5 μM of each primer, 2.5 units of Taq polymerase (BRL) and 5 μl of purified Grand fir stem cDNA library phage as template (1.5×10⁹pfu/ml). Analysis of the PCR reaction products by agarose gel electrophoresis (Sambrock, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) revealed that only the combination of primers C and D generated a specific PCR product of approximately 110 bps (base pairs). This PCR product was gel purified, ligated into pT7Blue (Novagen), and transformed into E. coli XL1-Blue cells. Plasmid DNA was prepared from 41 individual transformants and the inserts were sequenced (DyeDeoxy Terminator Cycle Sequencing, Applied Biosystems). Four different insert sequences were identified, and were designated as probes 1 (SEQ ID NO: 1 1), 2 (SEQ ID NO: 12), 4 (SEQ ID NO: 13) and 5 (SEQ ID NO: 14).
Subsequent isolation of four new cDNA species (AG1.28 (SEQ ID NO: 15); AG2.2 (SEQ ID NO: 1); AG4.30 (SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19)), encoding terpene synthases from Grand fir corresponding to probes 1 (SEQ ID NO: 11), 2 (SEQ ID NO: 12), 4 (SEQ ID NO: 13) and 5 (SEQ ID NO: 14), respectively, allowed the identification of three additional conserved sequence elements which were used to design a set of three new PCR primers: Primer E (5′-GGI GA(A/G) A(A/C)(A/G) (A/G)TI ATG GA(A/G) GA(A/G) GC-3′)(SEQ ID NO: 21); Primer F (5′-GA(A/G) (C/T)TI CA(G/A) (C/T)TI (A/C/T)(C/G/T)I (A/C)GI TGG TGG-3′)(SEQ ID NO: 22) and Primer G (5′-CCA (A/G)TT IA(A/G) ICC (C/T)TT IAC (A/G)TC-3′)(SEQ ID NO: 23). [0100]
Degenerate primer E (SEQ ID NO: 21) was designed to conserved element GE(K/T)(V/I)M(E/D)EA (SEQ ID NO: 26) and degenerate primer F (SEQ ID NO: 22) was designed to conserved element Q(F/Y/D)(I/L)(T/L/R)RWW (SEQ ID NO: 27) by comparing the sequences of five cloned terpene synthases from Grand fir: a monoterpene synthase corresponding to probe 2 (SEQ ID NO: 12), two sesquiterpene synthases corresponding to probe 4 (SEQ ID NO: 13) and probe 5 (SEQ ID NO: 14), respectively, a previously described diterpene synthase (Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996) [0101] J. Biol. Chem. 271:23262-23268), and a truncated terpene synthase corresponding to probe 1 (SEQ ID NO: 11). Degenerate primer G (SEQ ID NO: 23) was designed according to the amino acid sequence DVIKG(F/L)NW (SEQ ID NO: 28) obtained from a peptide generated by trypsin digestion of purified (−)-pinene synthase from Grand fir. Primers E (SEQ ID NO: 21) and F (SEQ ID NO: 22) were independently used for PCR amplification in combination with primer G (SEQ ID NO: 23), with Grand fir stem cDNA library as template. The combination of primers E (SEQ ID NO: 21) and G (SEQ ID NO: 23) yielded a specific PCR product of approximately 1020 bps. This PCR product was ligated into pT7Blue and transformed into E. coli XL1-Blue. Plasmid DNA was prepared from 20 individual transformants and inserts were sequenced from both ends. The sequence of this 1022 bp insert was identical for all 20 plasmids and was designated as probe 3 (SEQ ID NO: 24).

EXAMPLE 2

Screening a Wounded Grand fir Stem cDNA Library [0102]
For library screening, 100 ng of each probe was amplified by PCR, gel purified, randomly labeled with [α-[0103] ³²P]dATP (Feinberg, A. P., and Vogelstein, B. (1984) Anal. Biochem. 137:266-267), and used individually to screen replica filters of 10⁵plaques of the wound-induced Grand fir stem cDNA library plated on E. coli LE392. Hybridization with probes 1 (SEQ ID NO: 11), 2 (SEQ ID NO: 12), 4 (SEQ ID NO: 13) and 5 (SEQ ID NO: 14) was performed for 14 h at 65 ° C. in 3×SSPE and 0.1% SDS. Filters were washed three times for 10 min at 55 ° C. in 3×SSPE with 0.1% SDS and exposed for 12 h to Kodak XAR film at −70° C. All of the λZAPII clones yielding positive signals were purified through a second round of hybridization (probe 1 (SEQ ID NO: 11) gave 25 positives, probe 2 (SEQ ID NO: 12) gave 16 positives, probe 4 (SEQ ID NO: 13) gave 49 positives and probe 5 (SEQ ID NO: 14) gave 12 positives).
Hybridization with probe 3 (SEQ ID NO: 24) was performed as before, but the filters were washed three times for 10 min at 65° C. in 3×SSPE and 0.1% SDS before exposure. Approximately 400 λZAPII clones yielded strong positive signals, and 34 of these were purified through a second round of hybridization at 65° C. Approximately 400 additional clones yielded weak positive signals with probe 3 (SEQ ID NO: 24), and 18 of these were purified through a second round of hybridization for 20 h at 45° C. Purified λZAP II clones isolated using all five probes were in vivo excised as Bluescript II SK(−) phagemids and transformed into [0104] E. coli XLOLR according to the manufacturer's instructions (Stratagene). The size of each cDNA insert was determined by PCR using T3 (SEQ ID NO: 29) and T7 (SEQ ID NO: 30) promoter primers and selected inserts (>1.5 kb) were partially sequenced from both ends.

EXAMPLE 3

Grand fir Monoterpene Synthase cDNA Expression in [0105] E. coli and Enzyme Assays
Except for cDNA clones AG3.18 (SEQ ID NO: 3) and AG3.48 (SEQ ID NO: 31), all of the partially sequenced inserts were either truncated at the 5′-end, or were out of frame, or bore premature stop codons upstream of the presumptive methionine start codon. For the purpose of functional expression, a 2023 bp insert fragment, extending from nucleotides 75 to 2097 of the sequence set forth in SEQ ID NO: 1, and a 1911 bp insert fragment, extending from [0106] nucleotide 1 to nucleotide 1910 of the sequence set forth in SEQ ID NO: 3, were subcloned in frame into pGEX vectors (Pharmacia). A 2016 bp fragment extending from nucleotide 73 to nucleotide 2088 of the sequence set forth in SEQ ID NO: 5 was subcloned in frame into the pSBETa vector (Schenk, P. M., Baumann, S., Mattes, R., and Steinbiss, H. -H. (1995) Biotechniques 19, 196-200). To introduce suitable restriction sites for subcloning, fragments were amplified by PCR using primer combinations 2.2-BamHI (5′-CAA AGG GAT CCA GAA TGG CTC TGG-3′)(SEQ ID NO: 33) and 2.2-NotI (5′-AGT AAG CGG CCG CTT TTT AAT CAT ACC CAC-3′)(SEQ ID NO: 34) with pAG2.2 insert (SEQ ID NO: 1) as template, 3.18-EcoRI (5′-CTG CAG GAA TTC GGC ACG AGC-3′)(SEQ ID NO: 35) and 3.18-SmaI (5′-CAT AGC CCC GGG CAT AGA TTT GAG CTG-3′)(SEQ ID NO: 36) with pAG3.18 insert (SEQ ID NO: 3) as template, and 10-NdeI (5-GGC AGG AAC ATA TGG CTC TCC TTT CTA TCG-3′)(SEQ ID NO: 37) and 10-BamHI (5′-TCT AGA ACT AGT GGATCC CCC GGG CTG CAG-3′)(SEQ ID NO: 38) with pAG10 insert (SEQ ID NO: 5) as template.
PCR reactions were performed in volumes of 50 μl containing 20 mM Tris/HCl (pH 8.8), 10 mM KCl, 10 mM (NH[0107] ₄)₂SO₄, 2 mM MgSO₄, 0.1% Triton X-100, 5 μg bovine serum albumin (BSA), 200 μM of each dNTP, 0.1 μM of each primer, 2.5 units of recombinant Pfu polymerase (Stratagene) and 100 ng plasmid DNA with the following program: denaturation at 94° C., 1 min; annealing at 60° C., 1 min; extension at 72° C., 3.5 min; 35 cycles with final extension at 72° C., 5 min. The PCR products were purified by agarose gel electrophoresis and used as template for a secondary PCR amplification with the identical conditions in total volumes of 250 μl each. Products from this secondary amplification were digested with the above indicated restriction enzymes, purified by ultrafiltration and then ligated, respectively, into BamHI/NotI-digested pGEX-4T-2 to yield plasmid pGAG2.2, into EcoRI/SmaI-digested pGEX-4T-3 to yield plasmid pGAG3.18, and into NdeI/BamHI-digested pSBETa to yield plasmid pSBAG10; these plasmids were then transformed into E. coli XL1-Blue or E. coli BL21(DE3).
For expression, bacterial strains [0108] E. coli XLOLR/pAG3.18, E. coli XLOLR/pAG3.48, E. coli XL1-Blue/pGAG2.2, E. coli XL1-Blue/pGAG3.18, and E. coli BL21(DE3)/pSBAG10 were grown to A₆₀₀₌0.5 at 37° C. in 5 ml of LB medium (Sambrock, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) supplemented with 100 μg ampicillin/ml or 30 μg kanamycin/ml as determined by the vector. Cultures were then induced by addition of 1 mM isopropyl-1-thio-β-D-galactopyranoside and grown for another 12 h at 20° C. Cells were harvested by centrifugation (2000×g, 10 min) and resuspended in either 1 ml monoterpene synthase assay buffer [50 mM Tris/HCl (pH 7.5), 500 mM KCl, 1 mM MgCl₂, 5 mM dithiothreitol, 0.05% (w/v) NaHSO₃and 10% (v/v) glycerol], 1 ml sesquiterpene synthase assay buffer [10 mM dibasic potassium phosphate, 1.8 mM monobasic potassium phosphate (pH 7.3), 140 mM NaCl, 10 mM MgCl₂, 5 mM dithiothreitol, 0.05% (w/v) NaHSO₃and 10% (v/v) glycerol], or 1 mnl diterpene synthase assay buffer [30 mM Hepes (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, pH 7.2), 7.5 mM MgCl₂, 5 mM dithiothreitol, 10 μM MgCl₂, 0.05% (w/v) NaHSO₃and 10% (v/v) glycerol].
Cells were disrupted by sonication (Braun-Sonic 2000 with microprobe at maximum power for 15 seconds at 0-4° C.), the homogenates were cleared by centrifugation (18,000×g, 10 min), and 1 ml of the resulting supernatant was assayed for monoterpene synthase activity with 2.5 μM of [1-[0109] ³H]geranyl diphosphate, for sesquiterpene synthase activity with 3.5 μM [1-³H]farnesyl diphosphate, or for diterpene synthase activity with 5 μM [1-³H]geranylgeranyl diphosphate following standard protocols (Croteau, R., and Cane, D. E. (1985) Methods Enzymol. 110:383-405; LaFever, R. E., Stofer Vogel, B., and Croteau, R. (1994) Arch. Biochem. Biophys. 313:139-149; Dehal, S. S., and Croteau, R. (1988) Arch. Biochem. Biophys. 261:346-356). In the case of the monoterpene synthase and sesquiterpene synthase assays, the incubation mixture was overlaid with 1 ml pentane to trap volatile products. In all cases, after incubation at 31° C. for 2 h, the reaction mixture was extracted with pentane (3×1 ml) and the combined extract was passed through a 1.5 ml column of anhydrous MgSO₄and silica gel (Mallinckrodt 60 Å) to provide the terpene hydrocarbon fraction free of oxygenated metabolites. The columns were subsequently eluted with 3×1 ml of ether to collect any oxygenated products, and an aliquot of each fraction was taken for liquid scintillation counting to determine conversion rate.
Product Identification—To obtain sufficient product for analysis by radio-GLC (gas liquid chromatography), chiral capillary GLC and GLC-MS (mass spectrum/spectrometry), preparative-scale enzyme incubations were carried out. Thus, the enzyme was prepared from 50 ml of cultured bacterial cells by extraction with 3 ml of assay buffer as above, and the extracts were incubated with excess substrate overnight at31° C. The hydrocarbon fraction was isolated by elution through MgSO[0110] ₄-silica gel as before, and the pentane eluate was concentrated for evaluation by capillary radio-GLC as described (Croteau, R., and Satterwhite, D. M. (1990) J. Chromatogr. 500:349-354), by chiral column capillary GLC (Lewinsohn, E., Savage, T. J., Gijzen, M., and Croteau, R. (1993) Phytochem. Anal. 4:220-225), and by combined GLC-MS [Hewlett-Packard 6890 GC-MSD with cool (40° C.) on-column injection, detection via electron impact ionization (70 eV), He carrier at 0.7 psi., column: 0.25 mm i.d.×30 m fused silica with 0.25 μm film of 5 MS (Hewlett-Packard) programmed from 35° C. (5 min hold) to 230° C. at 5° C./min].

EXAMPLE 4

Sequence Analysis [0111]
Inserts of all recombinant bluescript plasmids and pGEX plasmids were completely sequenced on both strands via primer walking and nested deletions (Sambrock, J., Fritsch, E. F., and Maniatis, T. (1989) [0112] Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) using the DyeDeoxy Terminator Cycle Sequencing method (Applied Biosystems). Sequence analysis was done using the Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.

EXAMPLE 5

RNA Extraction and Northern Blotting [0113]
Grand fir sapling stem tissue was harvested prior to wounding or two days after wounding by a standard procedure (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) [0114] Arch. Biochem. Biophys. 289:267-273). Total RNA was isolated (Lewinsohn, E., Steele, C. L., and Croteau, R. (1994) Plant Mol. Biol. Rep. 12:20-25) and 20 μg of RNA per gel lane was separated under denaturing conditions (Sambrock, J., Fritsch, E. P., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and transferred to nitrocellulose membranes (Schleicher and Schuell) according to the manufacturer's protocol. To prepare hybridization probes, cDNA fragments of 1.4-1.5 kb were amplified by PCR from AG2.2 (SEQ ID NO: 1) with primer JB29 (5′-CTA CCA TTC CAA TAT CTG-3′)(SEQ ID NO: 39) and primer 2-8 (5′-GTT GGA TCT TAG AAG TTC CC-3′)(SEQ ID NO: 40), from AG3.18 (SEQ ID NO: 3) with primer 3-9 (5′-TTT CCA TTC CAA CCT CTG GG-3′)(SEQ ID NO: 41) and primer 3-11 (5′-CGT AAT GGA AAG CTC TGG CG-3′)(SEQ ID NO: 42), and from AG10 (SEQ ID NO: 5) with primer 7-1 (5′-CCT TAC ACG CCT TTG GAT GG-3′)(SEQ ID NO: 43) and primer 7-3 (5′-TCT GTT GAT CCA GGA TGG TC-3′)(SEQ ID NO: 44). The probes were randomly labeled with [α-³²P]dATP (Feinberg, A. P., and Vogelstein, B. (1984) Anal. Biochem. 137:266-267). Blots were hybridized for 24 h at 55° C. in 3×SSPE and 0.1% SDS, washed at 55° C. in 1×SSPE and 0.1%, SDS and subjected to autoradiography as described above at −80° C. for 24 h.

EXAMPLE 6

Cloning and Characterization of Clones AG1.28 (SEQ ID NO: 15). AG2.2 (SEQ ID NO: 1). AG4.30 (SEQ ID NO: 17) and AG 5.9 (SEQ ID NO: 19) [0115]
Similarity-Based Cloning of Grand fir Terpene Synthases—Grand fir has been developed as a model system for the study of induced oleoresin production in conifers in response to wounding and insect attack (Johnson, M. A., and Croteau, R. (1987) in [0116] Ecology and Metabolism of Plant Lipids (Fuller, G., and Nes, W. D., eds) pp. 76-91, American Chemical Society Symposium Series 325, Washington, D.C.; Gijzen, M., Lewinsohn, E., Savage, T. J., and Croteau, R. B. (1993) in Bioactive Volatile Compounds from Plants (Teranishi, R., Buttery, R. G., and Sugisawa, H., eds) pp. 8-22, American Chemical Society Symposium Series 525, Washington, D.C.; Raffa, K. F., and Berryman, A. A. (1982) Can. Entomol. 114:797-810; Steele, C., Lewinsohn, E., and Croteau, R. (1995) Proc. Natl. Acad. Sci. USA 92:4164-4168; Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) in Regulation of Isopentenoid Metabolism (Nes, W. D., Parish, E. J., and Trzaskos, J. M., eds) pp. 8-17, American Chemical Society Symposium Series 497, Washington, D.C.). The chemistry and biosynthesis of the oleoresin monoterpenes, sesquiterpenes and diterpenes have been well defined (Lewinsohn, E., Savage, T. J., Gijzen, M., and Croteau, R. (1993) Phytochem. Anal. 4:220-225; Lewinsohn, E., Gijzen, M., and Croteau, R. (1991) Plant Physiol. 96:44-49; Funk, C., Lewinsohn, E., Stofer Vogel, B., Steele C., and Croteau, R. (1994) Plant Physiol. 106:999-1005; Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289:267-273; Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173; LaFever, R. E., Stofer Vogel, B., and Croteau, R. (1994) Arch. Biochem. Biophys. 313:139-149; Funk, C., and Croteau, R. (1994) Arch. Biochem. Biophys. 308:258-266); however, structural analysis of the responsible terpene synthases as well as studies on the regulation of oleoresinosis require the isolation of cDNA species encoding the terpene synthases. Protein purification from conifers, as the basis for cDNA isolation, has been of limited success (Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268) and thus far has not permitted cloning of any of the monoterpene synthases from these species (Steele, C., Lewinsohn, E., and Croteau, R. (1995) Proc. Natl. Acad. Sci. USA 92:4164-4168).
As a possible alternative to protein-based cloning of terpene synthases, a homology-based PCR strategy was proposed (Steele, C., Lewinsohn, E., and Croteau, R. (1995) [0117] Proc. Natl. Acad. Sci. USA 92:4164-4168) that was founded upon the three terpene synthases of plant origin then available, a monoterpene synthase, (−)-4S-limonene synthase, from spearmint (Mentha spicata, Lamiaceae) (Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024), a sesquiterpene synthase, 5-epi-aristolochene synthase, from tobacco (Nicotiana tabacum, Solanaceae) (Facchini, P. J., and Chappell, J. (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092), and a diterpene synthase, casbene synthase, from castor bean (Ricinus communis, Euphorbiaceae) (Mau, C. J. D., and West, C. A. (1994) Proc. Natl. Acac Sci. USA 91:8497-8501). Despite the taxonomic distances between these three angiosperm species and the differences in substrate utilized, reaction mechanism and product type of the three enzymes, a comparison of the deduced amino acid sequences identified several conserved regions that appeared to be useful for the design of degenerate PCR primers (see Example 1). Using cDNA from a wound-induced Grand fir stem library as template, PCR primers C (SEQ ID NO: 9) and D (SEQ ID NO: 10) amplified products corresponding to four distinct sequence groups, all of which showed significant similarity to sequences of cloned terpene synthases of plant origin. The four different inserts were designated as probes 1 (SEQ ID NO: 11), 2 (SEQ ID NO: 12), 4 (SEQ ID NO: 13) and 5 (SEQ ID NO: 14), and were employed for isolation of the corresponding cDNA clones by plaque hybridization.
Screening of 10[0118] ⁵cDNA phage plaques from the wounded Grand fir stem library, with each of the four probes, yielded a four-fold difference in the number of positives, most likely reflecting different levels of expression of the corresponding genes. Size selected inserts (>1.5 kb) of purified and in vivo excised clones were partially sequenced from both ends, and were shown to segregate into four distinct groups corresponding to the four hybridization probes. Since all cDNAs corresponding to probes 1 (SEQ ID NO: 11), 4 (SEQ ID NO: 13) and 5 (SEQ ID NO: 14) were truncated at their 5′-ends, only inserts of the largest representatives of each group, clone AG1.28 (SEQ ID NO: 15), clone AG2.2 (SEQ ID NO: 1) (apparently full length), clone AG4.30 (SEQ ID NO: 17) and clone AG5.9 (SEQ ID NO: 19), were completely sequenced. Clone AG1.28 (SEQ ID NO: 15)(2424 bps) includes an open reading frame (ORF) of 2350 nucleotides (nts) encoding 782 amino acids (SEQ ID NO: 16); clone AG2.2 (SEQ ID NO: 1)(2196 bps), includes an ORF of 1881 nts encoding 627 amino acids (SEQ ID NO: 2); clone AG4.30 (SEQ ID NO: 17)(1967 bps) includes an ORF of 1731 nts encoding 577 amino acids (SEQ ID NO: 18) and clone AG5.9 (SEQ ID NO: 19)(1416 bps) includes an ORF of 1194 nucleotides encoding 398 amino acids (SEQ ID NO: 20).
cDNA clones AG1.28 (SEQ ID NO: 15), AG2.2 (SEQ ID NO: 1), AG4.30 (SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19) were compared pairwise with each other and with other cloned plant terpene synthases. Truncated clone AG1.28 (SEQ ID NO: 15) resembled most closely in size and sequence (72% similarity, 49% identity) a diterpene cyclase, abietadiene synthase, from Grand fir (Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996) [0119] J. Biol. Chem. 271:23262-23268). Clones AG4.30 (SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19) share approximately 80% similarity (60% identity) at the amino acid level, and are almost equally distant from both clone AG1.28 (SEQ ID NO: 15) and full-length clone AG2.2 (SEQ ID NO: 1)(range of 65-70% similarity and 45-47% identity); the amino acid sequence similarity between AG1.28 (SEQ ID NO: 15) and AG2.2 (SEQ ID NO: 1) is 65% (41% identity). Considering the high level of homology between AG4.30 (SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19), these comparisons suggest that the four new cDNAs, AG1.28 (SEQ ID NO: 15), AG2.2 (SEQ ID NO: 1), AG4.30 (SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19), represent the three major subfamilies of Grand fir terpene synthase genes encoding monoterpene synthases, sesquiterpene synthases and diterpene synthases.
Identification of cDNA Clone AG2.2 (SEQ ID NO: 1) as Myrcene Synthase—The pAG2.2 insert (SEQ ID NO: 1) appeared to be a full-length clone encoding a protein of molecular weight 72,478 with a calculated pI at 6.5. The size of the translated protein encoded by AG2.2 (SEQ ID NO: 1) (627 residues)(SEQ ID NO: 2) is in the range of the monoterpene synthase preproteins for limonene synthase from spearmint (Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993) [0120] J. Biol. Chem. 268:23016-23024) and Perilla frutescens (Yuba, A., Yazaki, K., Tabata, M., Honda, G., and Croteau, R. (1996) Arch. Biochem. Biophys. 332:280-287), but is about 240 amino acids shorter than the two gymnosperm diterpene synthase preproteins for abietadiene synthase (Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268) and taxadiene synthase (Wildung, M. R., and Croteau, R. (1996) J. Biol. Chem. 271:9201-9204). Monoterpene and diterpene biosynthesis are compartmentalized in plastids whereas sesquiterpene biosynthesis is cytosolic (reviewed in Kleinig, H. (1989) Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:39-53; Gershenzon, J., and Croteau, R. (1993) in Lipid Metabolism in Plants (Moore, T. S. Jr., ed) pp. 339-388, CRC Press, Boca Raton, Fla.; McGarvey, D. J., and Croteau, R. (1995) Plant Cell 7, 1015-1026); thus, monoterpene and diterpene synthases are encoded as preproteins bearing an amino-terminal transit peptide for import of these nuclear gene products into plastids where they are proteolytically processed to the mature forms (Keegstra, K., Olsen, J. J., and Theg, S. M. (1989) Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:471-501; von Heijne, G., Stepphuhn, J., and Herrmann (1989) Eur. J. Biochem. 180:535-545). Both the size of the deduced protein and the presence of an N-terminal domain (of 60 to 70 amino acids) with features characteristic of a targeting sequence [rich in serine residues (16-18%) and low in acidic residues (four Asp or Glu) (Keegstra, K., Olsen, J. J., and Theg, S. M. (1989) Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:471-501; von Heijne, G., Stepphuhn, J., and Herrmann (1989) Eur. J. Biochem. 180:535-545)] suggest that AG2.2 (SEQ ID NO: 1) encodes a monoterpene synthase rather than a sesquiterpene synthase or a diterpene synthase.
Since pAG2.2 contained the terpene synthase insert in reversed orientation, the ORF was subcloned in frame with glutathione S-transferase, for ultimate ease of purification (Bohlmann, J., DeLuca, V., Eilert, U., and Martin, W. (1995) [0121] Plant J. 7:491-501; Bohlmann, J., Lins, T., Martin, W., and Eilert, U. (1996) Plant Physiol. 111:507-514), into pGEX-4T-2, yielding plasmid pGAG2.2. The recombinant fusion protein was expressed in E. coli strain XL1-Blue/pGAG2.2, then extracted and assayed for monoterpene synthase, sesquiterpene synthase and diterpene synthase activity using tritium labeled geranyl diphosphate, farnesyl diphosphate and geranylgeranyl diphosphate as the respective substrate. Enzymatic production of a terpene olefin was observed only with geranyl diphosphate as substrate, and the only product was shown to be myrcene by radio-GLC and GLC-MS comparison to an authentic standard (FIG. 3). Bacteria transformed with pGEX vector containing the AG2.2 insert (SEQ ID NO: 1) in antisense orientation did not afford detectable myrcene synthase activity when induced, and the protein isolated and assayed as above. A myrcene synthase cDNA has not been obtained previously from any source, although myrcene is a minor co-product (2%) of the native and recombinant limonene synthase from spearmint (Rajaonarivony, J. I. M., Gershenzon, J., and Croteau, R. (1992) Arch. Biochem. Biophys. 296:49-57; Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024) and of several enzymes from sage (Croteau, R., and Satterwhite, D. M. (1989) J. Biol. Chem. 264:15309-15315). cDNA cloning and functional expression of myrcene synthase, which is one of several wound-inducible monoterpene synthase activities of Grand fir (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289:267-273), demonstrates that this acyclic monoterpene is formed by a distinct enzyme and is not a co-product of another synthase.

EXAMPLE 7

Cloning and Characterization of Clones AG3.18 (SEQ ID NO: 3) Encoding (−)-Pinene Synthase and cDNA Clone AG10 (SEQ ID NO: 5) Encoding (−)-Limonene Synthase [0122]
Identification of cDNA Clone AG3.18 (SEQ ID NO: 3) as (−)-Pinene Synthase and cDNA Clone AG10 (SEQ ID NO: 5) as (−)-Limonene Synthase—Alignment of the four new terpene synthase EDNA sequences (AG1.28 (SEQ ID NO: 15), AG2.2 (SEQ ID NO: 1), AG4.30 (SEQ ID NO: 17) and AG.5.9 (SEQ ID NO: 19)), and that for abietadiene synthase (Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996) [0123] J. Biol. Chem. 271:23262-23268), allowed the identification of several conserved sequence motifs among this enzyme family from Grand fir, which provided the foundation for an extended similarity-based cloning approach. Two new sense primers E (SEQ ID NO: 21) and F (SEQ ID NO: 22) were designed according to conserved sequence elements, whereas a degenerate antisense primer G (SEQ ID NO: 23) was designed based upon very limited amino acid sequence information from pinene synthase (see Example 1). Only the combination of primers E (SEQ ID NO: 21) and G (SEQ ID NO: 23) amplified a specific product of 533 bps, which was designated as probe 3 (SEQ ID NO: 24).
Hybridization of 10[0124] ⁵Grand fir ZAP II cDNA clones with probe 3 (SEQ ID NO: 24) yielded two types of signals comprised of about 400 strongly positive clones and an equal number of weak positives, indicating that the probe recognized more than one type of cDNA. Thirty-four of the former clones and 18 of the latter were purified, the inserts were selected by size (2.0-2.5 kb), and the in vivo excised clones were partially sequenced from both ends. Those clones which afforded weak hybridization signals were shown to contain inserts that were either identical to myrcene synthase clone AG2.2 (SEQ ID NO: 1) or exhibited no significant sequence similarity to terpene synthases. Clone AG3.48 (SEQ ID NO: 31) contained the myrcene synthase ORF in the correct orientation and in frame for expression from the Bluescript plasmid vector. This cDNA was functionally expressed in E. coli and the resulting enzyme was shown to accept only geranyl diphosphate as the prenyl diphosphate substrate and to produce myrcene as the exclusive reaction product. This finding with AG3.48 (SEQ ID NO: 31) confirms that expression of AG2.2 (SEQ ID NO: 1) as the glutathione S-transferase fusion protein from pGAG2.2 does not influence substrate utilization or product outcome of the myrcene synthase.
Clones which gave strong hybridization signals segregated into distinct sequence groups represented by clone AG3.18 (SEQ ID NO: 3)(2018 bp insert with ORF of 1884 nt; encoded protein of 628 residues at 71,505 Da and pI of 5.5) and AG10 (SEQ ID NO: 5)(2089 bp insert with ORF of 1911 nt; encoded protein of 637 residues at 73,477 Da and pI of 6.4). AG3.18 (SEQ ID NO: 3) and AG10 (SEQ ID NO: 5) form a subfamily together with the myrcene synthase clone AG2.2 (SEQ ID NO: 1) that is characterized by a minimum of 79% pairwise similarity (64% identity) at the amino acid level. Like myrcene synthase, both AG3.18 (SEQ ID NO: 3) and AG10 (SEQ ID NO: 5) encode N-terminal sequences of 60 to 70 amino acids which are rich in serine (19-22% and 11-15%, respectively) and low in acidic residues (4 and 2, respectively) characteristic of plastid transit peptides (Keegstra, K., Olsen, J. J., and Theg, S. M. (1989) [0125] Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:471-501; von Heijne, G., Stepphuhn, J., and Herrmann (1989) Eur. J. Biochem. 180:535-545).
Plasmid pAG3.18 (SEQ ID NO: 3) contained the presumptive terpene synthase ORF in frame for direct expression from the bluescript plasmid, whereas the AG10 (SEQ ID NO: 5) ORF was in reversed orientation. Both AG3.18 (SEQ ID NO: 3) and AG10 (SEQ ID NO: 5) were subcloned into expression vectors yielding plasmids pGAG3.18 and pSBAG10. Recombinant proteins were expressed in bacterial strain [0126] E. coli XLOLR/pAG3.18, E. coli XL1-Blue/pGAG3.18 and E. coli BL21(DE3)/pSBAG10. When extracts of the induced cells were tested for terpene synthase activity with all of the potential prenyl diphosphate substrates, only geranyl diphosphate was utilized. Extracts from E. coli BL21(DE3)/pSBAG10 converted geranyl diphosphate to limonene as the major product with lesser amounts of α-pinene, β-pinene and β-phellandrene, as determined by radio-GLC and combined GLC-MS (FIG. 5). Chiral phase capillary GLC on β-cyclodextrin revealed the limonene product to be the (−)-4S-enantiomer and the pinene products to be the related (−)-(1S:5S)-enantiomers. Although optically pure standards were not available for the analysis, stereochemical considerations suggest that the minor product β-phellandrene is also the mechanistically related (−)-(4S)-antipode (Gambliel, H., and Croteau, R. (1984) J. Biol. Chem. 259:740-748; Croteau, R., Satterwhite, D. M., Cane, D. E., and Chang, C. C. (1988) J. Biol. Chem. 263:10063-10071; Wagschal, K., Savage, T. J., and Croteau, R. (1991) Tetrahedron 47:5933-5944; Croteau, R., Satterwhite, D. M., Wheeler, C. J., and Felton, N. M. (1989) J. Biol. Chem. 264:2075-2080; LaFever, R. E., and Croteau, R. (1993) Arch. Biochem. Biophys. 301:361-366). Similar analysis of the monoterpene products generated from geranyl diphosphate by cell-free extracts of E. coli XLOLR/pAG3.18 and E. coli XL1-Blue/pGAG3.18 demonstrated the presence of a 42:58% mixture of α-pinene and β-pinene (FIG. 4), the same product ratio previously described for the purified, native (−)-pinene synthase from Grand fir (Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173). Chiral phase capillary GLC confirmed the products of the recombinant pinene synthase to be the (−)-(1S:5S)-enantiomers, as expected. No other monoterpene co-products were detected with the recombinant (−)-pinene synthase, as observed previously for the native enzyme (Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173).
Evidence for the formation of both α- and β-pinene by a single enzyme has been previously provided through co-purification studies, and differential inhibition and inactivation studies, as well as by isotopically sensitive branching experiments (Gambliel, H., and Croteau, R. (1984) [0127] J. Biol. Chem. 259:740-748; Wagschal, K. C., Pyun, H. -J., Coates, R. M., and Croteau, R. (1994) Arch. Biochem. Biophys. 308:477-487; Wagschal, K., Savage, T. J., and Croteau, R. (1991) Tetrahedron 47:5933-5944; Croteau, R., Wheeler, C. J., Cane, D. E., Ebert, R., and Ha, H. -J. (1987) Biochemistry 26:5383-5389). The cDNA cloning of pinene synthase provides the ultimate proof that a single enzyme forms both products. The calculated molecular weight of the (−)-pinene synthase deduced from AG3.18 (SEQ ID NO: 3) is approximately 64,000 (excluding the putative transit peptide), which agrees well with the molecular weight of 63,000 established for the native enzyme from Grand fir by gel permeation chromatography and SDS-PAGE (Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173).
A limonene synthase cDNA has thus far been cloned only from two very closely related angiosperm species (Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993) [0128] J. Biol. Chem. 268:23016-23024; Yuba, A., Yazaki, K., Tabata, M., Honda, G., and Croteau, R. (1996) Arch. Biochem. Biophys. 332:280-287), and the isolation of a pinene synthase cDNA has not been reported before. Pinene synthase has previously received considerable attention as a major defense-related monoterpene synthase in conifers (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289:267-273; Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173). In the Grand fir cDNA library, which was synthesized from mRNA obtained from wound-induced sapling stems, clones corresponding to pinene synthase are at least ten times more abundant than clones for myrcene synthase. This finding reflects the relative proportions of the induced levels of activities of these enzymes in Grand fir saplings; pinene synthase and limonene synthase are the major monoterpene synthase activities whereas the induced level of myrcene synthase activity is relatively low (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289:267-273). The cDNAs for inducible monoterpene synthases provide probes for genetic and molecular analysis of oleoresin-based defense in conifers. Northern blots (FIG. 6) of total RNA extracted from non-wounded sapling stems and from stems two days after wounding (when enzyme activity first appears) were probed with cDNA fragments for AG2.2 (SEQ ID NO: 1), AG3.18 (SEQ ID NO: 3) and AG10 (SEQ ID NO: 5), and thus demonstrated that increased mRNA accumulation for monoterpene synthases is responsible for this induced, defensive response in Grand fir. The availability of cloned, defense-related monoterpene synthases presents several possible avenues for transgenic manipulation of oleoresin composition to improve tree resistance to bark beetles and other pests. For example, altering the monoterpene content of oleoresin may chemically disguise the host and decrease insect aggregation by changing the levels of pheromone precursors or predator attractants, or lower infestation by increasing toxicity toward beetles and their pathogenic fungal associates (Johnson, M. A., and Croteau, R. (1987) in Ecology and Metabolism of Plant Lipids (Fuller, G., and Nes, W. D., eds) pp. 76-91, American Chemical Society Symposium Series 325, Washington, D.C.; Gijzen, M., Lewinsohn, E., Savage, T. J., and Croteau, R. B. (1993) in Bioactive Volatile Compounds from Plants (Teranishi, R., Buttery, R. G., and Sugisawa, H., eds) pp. 8-22, American Chemical Society Symposium Series 525, Washington, D.C.; Byers, J. A. (1995) in Chemical Ecology of Insects 2 (Carde, R. T., and Bell, W. J., eds) pp. 154-213, Chapman and Hall, New York).

EXAMPLE 8

Properties of the Recombinant Monoterpene Synthases Encoded by cDNA Clones AG2.2 (SEQ ID NO: 1). AG3.18 (SEQ ID NO: 3) and AG10 (SEQ ID NO: 5) [0129]
All three recombinant enzymes require Mn[0130] ²⁺ for activity, and Mg²⁺ is essentially ineffective as the divalent metal ion cofactor. This finding confirms earlier results obtained with the native monoterpene synthases of Grand fir and lodgepole pine (Pinus contorta) (Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173; Savage, T. J., Hatch, M. W., and Croteau, R. (1994) J. Biol. Chem. 269:4012-4020). All terpene synthases and phenyltransferases are thought to employ a divalent metal ion, usually Mg²⁺ or Mn²⁺, in the ionization steps of the reaction sequence to neutralize the negative charge of the diphosphate leaving group (Croteau, R. (1987) Chem. Rev. 87:929-954; Cane, D. E. (1992) Ciba Found Symp. Ser. 171:163-167; Poulter, C. D., and Rilling, H. C. (1981) in Biosynthesis of Isoprenoid Compounds (Porter, J. W., and Spurgeon, S. L., eds) Vol. 1, pp. 161-224, Wiley & Sons, New York), and all relevant sequences thus far obtained bear a conserved aspartate rich element (DDXXD)(SEQ ID NO: 45) considered to be involved in divalent metal ion binding (Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268; Ashby, M. N., and Edwards, P. A. (1990) J. Biol. Chem. 265:13157-13164; Chen, A., Kroon, P. A., and Poulter, D. C. (1994) Protein Sci. 3:600-607; Tarshis, L. C., Yan, M., Poulter, C. D., and Sacchettini, J. C. (1994) Biochemistry 33:10871-10877; Cane, D. E., Sohng, J. K., Lamberson, C. R., Rudnicki, S. M., Wu, Z., Lloyd, M. D., Oliver, J. S., and Hubbard, B. R. (1994) Biochemistry 33:5846-5857; Proctor, R. H., and Hohn, T. M. (1993) J. Biol. Chem. 268:4543-4548). In addition to this strict, general dependence on a divalent metal ion, the monoterpene synthases of conifers are unique in their further requirement for a monovalent cation (K⁺), a feature that distinguishes the gymnosperm monoterpene synthases from their counterparts from angiosperm species and implies a fundamental structural and/or mechanistic difference between these two families of catalysts (Savage, T. J., Hatch, M. W., and Croteau, R. (1994) J. Biol. Chem. 269:4012-4020). All three recombinant monoterpene synthases depend upon K⁺, with maximum activity achieved at approximately 500 mM KCl. A requirement for K⁺ has been reported for a number of different types of enzymes, including those that catalyze phosphoryl cleavage or transfer reactions (Suelter, C. H. (1970) Science 168:789-794) such as Hsc70 ATPase (Wilbanks, S. M., and McKay, D. B. (1995) J. Biol Chem. 270:2251-2257). The crystal structure of bovine Hsc70 ATPase indicates that both Mg²⁺ and K⁺ interact directly with phosphate groups of the substrate and implicates three active site aspartate residues in Mg²⁺ and K⁺ binding (Wilbanks, S. M., and McKay, D. B. (1995) J. Biol. Chem. 270:2251-2257), reminiscent of the proposed role of the conserved DDXXD (SEQ ID NO: 45) motif of the terpene synthases and phenyltransferases in divalent cation binding, a function also supported by recent site directed mutagenesis (Marrero, P. F., Poulter, C. D., and Edwards, P. A. (1992) J. Biol. Chem. 267:21873-21878; Joly, A., and Edwards, P. A. (1993) J. Biol. Chem. 268: 26983-26989; Song, L., and Poulter, C. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:3044-3048; Koyama, T., Tajima, M., Sano, H., Doi, T., Koike-Takeshita, A., Obata, S., Nishino, T., and Ogura, K. (1996) Biochemistry 35:9533-9538) and by X-ray structural analysis (Tarshis, L. C., Yan, M., Poulter, C. D., and Sacchettini, J. C. (1994) Biochemistry 33:10871-10877) of farnesyl diphosphate synthase.
cDNA cloning and functional expression of the myrcene, limonene and pinene synthases from Grand fir represent the first example of the isolation of multiple synthase genes from the same species, and provide tools for evaluation of structure-function relationships in the construction of acyclic, monocyclic and bicyclic monoterpene products and for detailed comparison to catalysts from phylogenetically distant plants that carry out ostensibly identical reactions (Gambliel, H., and Croteau, R. (1984) [0131] J. Biol. Chem. 259:740-748; Rajaonarivony, J. I. M., Gershenzon, J., and Croteau, R. (1992) Arch. Biochem. Biophys. 296:49-57; Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024; Adam, K. -P., Crock, J., and Croteau, R. (1996) Arch. Biochem. Biophys. 332:352-356). The recent acquisition of cDNA isolates encoding sesquiterpene synthases and diterpene synthases (Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268) from Grand fir should, together with the monoterpene synthases, also permit addressing the structural basis of chain-length specificity for prenyl diphosphate substrates in this family of related enzymes.

EXAMPLE 9

Sequence Comparison of Certain Cloned Monoterpene Synthases [0132]
Previous studies based on substrate protection from inactivation with selective amino acid modifying reagents have implicated functionally important cysteine, histidine and arginine residues in a range of different monoterpene synthases (Rajaonarivony, J. I. M., Gershenzon, J., and Croteau, R. (1992) [0133] Arch. Biochem. Biophys. 296:49-57; Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173; Savage, T. J., Hatch, M. W., and Croteau, R. (1994) J. Biol. Chem. 269:4012-4020; Rajaonarivony, J. I. M., Gershenzon, J., Miyazaki, J., and Croteau, R. (1992) Arch. Biochem. Biophys. 299:77-82; Savage, T. J., Ichii, H., Hume, S. D., Little, D. B., and Croteau, R. (1994) Arch. Biochem. Biophys. 320:257-265). Sequence alignment of 21 terpene synthases of plant origin (Colby, S. M., Alonso, W. R., Katahira, E. J., McGarvey, D. J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024; Stofer Vogel, B., Wildung, M. R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268; Facchini, P. J., and Chappell, J. (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092; Mau, C. J. D., and West, C. A. (1994) Proc. Natl. Acad. Sci. USA 91:8497-8501; Yuba, A., Yazaki, K., Tabata, M., Honda, G., and Croteau, R. (1996) Arch. Biochem. Biophys. 332:280-287; Wildung, M. R., and Croteau, R. (1996) J. Biol. Chem. 271:9201-9204; Yamaguchi, S., Saito, T., Abe, H., Yamane, H., Murofuishi, N., and Kamiya, Y. (1996) Plant J. 10:203-213; Dudareva, N., Cseke, L., Blanc, V. M., and Pichersky, E. (1996) Plant Cell 8:1137-1148; Chen, X. -Y., Chen, Y., Heinstein, P., and Davisson, V. J. (1995) Arch. Biochem. Biophys. 324:255-266; Chen, X. -Y., Wang, M., Chen, Y., Davisson, J., and Heinstein, P. (1996) J. Nat. Prod. 59:944-951; Back, K., and Chappell, J. (1995) J. Biol Chem. 270:7375-7381) reveals two absolutely conserved arginine residues, corresponding to Arg¹⁸⁴and Arg³⁶⁵of pinene synthase (SEQ ID NO: 4), one highly conserved cysteine residue (pinene synthase Cys⁵⁴³)(SEQ ID NO: 4), and one highly conserved histidine residue (pinene synthase His¹⁸⁶)(SEQ ID NO: 4). The DDXXD (SEQ ID NO: 45) sequence motif (pinene synthase Asp³⁷⁹, Asp³⁸⁰and Asp³⁸³) (SEQ ID NO: 4) is absolutely conserved in all relevant plant terpene synthases, as are several other amino acid residues corresponding to Phe¹⁹⁸, Leu²⁴⁸, Glu³²², Trp³²⁹, Trp⁴⁶⁰and Pro⁴⁶⁷of pinene synthase (SEQ ID NO: 4).
Amino acid sequences of the plant terpene synthases were compared with each other and with the deduced sequences of several sesquiterpene synthases cloned from microorganisms (Proctor, R. H., and Hohn, T. M. (1993) [0134] J. Biol. Chem. 268:4543-4548; Back, K., and Chappell, J. (1995) J. Biol. Chem. 270:7375-7381; Hohn, T. M., and Desjardins, A. E. (1992) Mol. Plant-Microbe Interactions 5:249-256). As with all other plant terpene synthases, no significant conservation in primary sequence exists between the monoterpene synthases from Grand fir and the terpene synthases of microbial origin, except for the DDXXD (SEQ ID NO: 45) sequence motif previously identified as a common element of all terpene synthases, and phenyltransferases which employ a related electrophilic reaction mechanism (Croteau, R., Wheeler, C. J., Cane, D. E., Ebert, R., and Ha, H. -J. (1987) Biochemistry 26:5383-5389; Chen, A., Kroon, P. A., and Poulter, D. C. (1994) Protein Sci. 3:600-607; McCaskill, D., and Croteau, R. (1997) Adv. Biochem. Engineering Biotech. 55:108-146). The evidence is presently insufficient to determine whether extant plant and microbial terpene synthases represent divergent evolution from a common ancestor, which may also have given rise to the phenyltransferases, or whether these similar catalysts evolved convergently.

EXAMPLE 10

A Strategy For Cloning Certain Gymnosperm Monoterpene Synthases [0135]

The present invention includes myrcene synthase, (−)-limonene synthase and (−)-pinene synthase proteins, and nucleic acid molecules that encode myrcene synthase, (−)-limonene synthase and (−)-pinene synthase proteins. The amino acid sequence of each of the myrcene synthase, (−)-limonene synthase and (−)-pinene synthase proteins of the present invention each includes at least one of the amino acid sequence elements disclosed in Table 1.

TABLE 1


		Orien-
Amino Acids	Sequence	tation	Comments

1.	70-77	H S N (L, I, V) W D D D	F only	HS makes a poor
		(SEQ ID NO: 46)		reverse primer
2.	148-153	A L D Y V Y	F and R
		(SEQ ID NO: 47)
3.	306-312	E L A K L E F	F and R
		(SEQ ID NO: 48)
4.	328-333	R W W K E S	F and R	F primer uses
		(SEQ ID NO: 49)		1^stnt of Ser
				codon; R uses
				1^sttwo nts
				of Arg codon
				(rare only)
5.	377-383	(V, I, L) L D D M Y D	F and R
		(SEQ ID NO: 50)
6.	377-383	(V, I, L) L D D L Y D	F and R	Degeneracy
		(SEQ ID NO: 51)		of V/I/L at 377
				too high for
				single primer
7.	377-383	(V, I, L) L D D I Y D	F and R
		(SEQ ID NO: 52)
8.	543-549	C Y M K D (N, H) P	R	F primer can
		(SEQ ID NO: 53)		also be
				constructed with
				this peptide but
				is too close to
				the 3′ end
				to be useful

The numbers set forth in Table 1 for the first and last amino acid residue of each of the peptide sequences is the number of the corresponding amino acid residue in the amino acid sequence of the (−)-pinene synthase (SEQ ID NO: 4) isolated from [0137] Abies grandis. Where a sequence of amino acid residues appears in brackets, e.g., (L,I,V) in Table 1, the first amino acid residue within the brackets is the residue that appears in the (−)-pinene synthase amino acid sequence set forth in SEQ ID NO: 4. The subsequent amino acid residues within the brackets represent other amino acid residues that commonly occur at the corresponding position in the amino acid sequence of other Abies grandis enzymes involved in terpene synthesis.
In Table 1, the letter “F” refers to the forward PCR reaction, i.e., the PCR reaction which synthesizes the sense nucleic acid strand that encodes a gymnosperm monoterpene synthase. The letter “R” refers to the reverse PCR reaction, i.e., the PCR reaction that synthesizes the antisense nucleic acid molecule that is complementary to the sense nucleic acid strand synthesized in the forward PCR reaction. [0138]
In order to clone nucleic acid molecules encoding myrcene synthase, (−)-limonene synthase and (−)-pinene synthase of the present invention, one or more oligonucleotide molecules corresponding to at least a portion of one of the amino acid sequences set forth in Table 1 can be used as a probe or probes with which to screen a genomic or cDNA library derived from one or more gymnosperm species. In this context, the term “corresponding,” or “correspond” or “corresponds,” means that the oligonucleotide base sequence either a) encodes all or part of at least one of the amino acid sequences set forth in Table 1, or b) is complementary to a base sequence that encodes all or part of at least one of the amino acid sequences set forth in Table 1. The oligonucleotide probe(s) may contain a synthetic base, such as inosine, which can be substituted for one or more of the four, naturally-occurring bases, i.e., adenine (“A”), guanine (“G”), cytosine (“C”) and thymine (“T”). Thus, for example, the following oligonucleotide sequences “correspond” to the tripeptide sequence M M M: 5′ATGATGATG3′ (sense orientation) (SEQ ID NO: 54); 3′TACTACTAC5′ (antisense orientation) (SEQ ID NO: 55) and 3′IACIACIAC5′ (SEQ ID NO: 56). [0139]
One or more oligonucleotide sequence(s), corresponding to at least a portion of at least one of the amino acid sequences set forth in Table 1, can be used to screen a nucleic acid library in order to identify myrcene synthase, (−)-limonene synthase and (−)-pinene synthase clones of the present invention, according to methods well known to one of ordinary skill in the art. See, e.g., Sambrook et al, supra. The stringency of the hybridization and wash conditions during library screening in accordance with the present invention, utilizing one or more oligonucleotide sequence(s) corresponding to at least a portion of at least one of the amino acid sequences set forth in Table 1, is at least: for the hybridization step, 6×SSPE, 40-45° C., for 36 hours; for the wash step, 3×SSPE, 45° C., 3×15 minute washes. The presently preferred hybridization and wash conditions during library screening, utilizing one or more oligonucleotide sequence(s) corresponding to at least a portion of at least one of the amino acid sequences set forth in Table 1, in accordance with the present invention are: for the hybridization step, 6×SSPE, 40-45° C., for 36 hours; for the wash step, 0.1×SSPE, 65° C.-70° C., 3×15 minute washes. [0140]

Examples of oligonucleotide sequences, corresponding to at least one of the amino acid sequences set forth in Table 1, that hybridize, under the foregoing hybridization and wash conditions, to the sense strands of the nucleic acid sequences of the present invention that encode myrcene synthase, (−)-limonene synthase or (−)-pinene synthase proteins are set forth in Table 2.

TABLE 2


Nucleic Acid Sequence	Corresponds to:

GTG TCG TTG GAG ACC CTG CTG CTG	SEQ ID No.46
(SEQ ID NO:57)

CGG GAG CTG ATG CAG ATG	SEQ ID No.47
(SEQ ID NO:58)

CTC GAG CGG TTC GAG CTC AAG	SEQ ID No.48
(SEQ ID NO:59)

GCC ACC ACC TTC CTC TCG	SEQ ID No.49
(SEQ ID NO:60)

GAG GAG CTG CTG TAC ATG CTG	SEQ ID No.50
(SEQ ID NO:61

GAG GAG CTG CTG GAG ATG CTG	SEQ ID No.51
(SEQ ID NO:62)

Similarly, each of the myrcene synthase, (−)-limonene synthase and (−)-pinene synthase clones set forth in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, or a portion thereof, may be used as a probe to screen a nucleic acid library in order to isolate monoterpene synthase clones of the present invention, according to methods well known to one of ordinary skill in the art. See, e.g., Sambrook et al, supra. The stringency of the hybridization and wash conditions during library screening in accordance with the present invention is at least: for the hybridization step, 6×SSPE buffer at 45° C. to 50° C. for 36 hours; for the wash step, 3×SSPE buffer at 50° C. (three, fifteen minute washes). In accordance with the present invention, the presently preferred hybridization and wash conditions during library screening utilizing any of the gymnosperm monoterpene synthase clones set forth in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, or a portion thereof, as probe are: for the hybridization step, 6×SSPE, 40-45° C., for 36 hours; for the wash step, 0.1×SSPE, 70° C.-75° C., 3×15 minute washes. [0142]
Additionally, at least two oligonucleotide sequence(s), each corresponding to at least a portion of at least one of the amino acid sequences set forth in Table 1, can be used in a PCR reaction to generate a portion of a myrcene synthase, (−)-limonene synthase or (−)-pinene synthase clone of the present invention, which can be used as a probe to isolate a full-length clone of a myrcene synthase, (−)-limonene synthase or (−)-pinene synthase clone of the present invention. Thus, oligonucleotides that are useful as probes in the forward PCR reaction correspond to at least a portion of at least one of the amino acid sequences disclosed in Table 1 as having the “F” orientation. Conversely, oligonucleotides that are useful as probes in the reverse PCR reaction correspond to at least a portion of at least one of the amino acid sequences disclosed in Table 1 as having the “R” orientation. PCR reactions can be carried out according to art-recognized PCR reaction conditions, such as the PCR reaction conditions set forth in Example 1 herein and as set forth in “PCR Strategies”, M. A. Innis, D. H. Gelfand and J. J. Sninsky, eds., 1995, Academic Press, San Diego, Calif. (Chapter 14); “PCR Protocols: A Guide to Methods and Applications”, M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White, eds., Academic Press, NY (1990). The presently preferred PCR reaction conditions are: [0143]

dNTPs 200 μM each

MgCl₂ 5-7 mM

F and R primers 100 nM - 1 μM each

Taq polymerase 1-2 units/reaction

cDNA template 10-100 ng/reaction

Buffers, PCR grade water, and Chill-out wax or mineral oil

The presently preferred thermocycler conditions are:



Denaturation	94° × 2 min	1 cycle
Denaturation	94° × 45 s	35 cycles
Annealing	42°-55° × 45 s - 1 min	″
Polymerization	72° × 1-2 min	″
Adenylation	72° × 10 min	1 cycle

EXAMPLE 11

Cloning and Characterization of cDNA Clones Encoding (−)-Camphene Synthase (−)-β-Phellandrene Synthase, Terpinolene Synthase and (−)-limonene/(−)-α-pinene Synthase [0145]
Comparison of resin analysis and the products generated by in vitro assay of the corresponding native enzymes (Lewinsohn, E., Savage, T. J., Gijzen, M., and Croteau, R. (1993) [0146] Phytochem. Anal. 4, 220-225; Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289, 267-273.), with the products of the available recombinant monoterpene synthases (Bohlmann, J., Steele, C. L., and Croteau, R. (1997) J. Biol. Chem. 272, 21784-21792), reveals that myrcene synthase, limonene synthase and pinene synthase do not account for all of the monoterpene synthases of grand fir. Molecular cloning of the complete family of grand fir monoterpene synthases is essential for evaluation of the regulatory role of each gene in constitutive and induced resin formation, can lead to the identification of useful genetic markers for resistance, provide the tools for engineering improved defense, and yield a more diverse set of recombinant catalysts for comparative mechanistic and structural study. Consequently, cDNA molecules encoding additional monoterpene synthases were isolated from a Grand fir cDNA library as described in this Example.
Additional Grand fir monoterpene synthases were cloned and analyzed as follows. [0147]
Substrates, Reagents and cDNA library. [1-[0148] ³H]Geranyl diphosphate (250 Ci/mol) (Croteau, R., Alonso, W. R., Koepp, A E., and Johnson, M. A. (1994) Arch. Biochem. Biophys. 309, 184-192), [1-³H]farnesyl diphosphate (125 Ci/mol) (Dehal, S. S., and Croteau, R. (1988) Arch. Biochem. Biophys. 261, 346-356) and [1-³H]geranylgeranyl diphosphate (120 Ci/mol) (LaFever, R. E., Stofer Vogel, B., and Croteau, R. (1994) Arch. Biochem. Biophys. 313, 139-149) were prepared as described in the foregoing publications. Terpenoid standards were from the inventors' own collection. All other biochemicals and reagents were purchased from Sigma Chemical Co. or Aldrich Chemical Co., unless otherwise noted. Construction of the λZAP II cDNA library, using mRNA isolated from wounded grand fir sapling stems (Lewinsohn, E., Steele, C. L., and Croteau, R. (1994) Plant Mol. Biol. Rep. 12, 20-25), was described previously in (Stofer Vogel, B., Wildung, M., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271, 23262-23268).
PCR-based probe generation. PCR was performed in a total buffer volume of 50 μl containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 5 mM MgCl[0149] ₂, 200 μM of each dNTP, 2.5 units of Tag polymerase (BRL) and 5 μl of purified grand fir stem cDNA library phage as template (1.5×10⁹pfu/ml). Three different PCR mixtures were evaluated containing either 1-5 μM of each primer, 1-5 μM of primer E (SEQ ID NO: 21) only, or 1-5 μM of primer G (SEQ ID NO: 23) only. After a denaturing step at 94° C. for 2 min, 35 cycles of amplification were performed employing the following temperature program using a Gradient 96 Robocycler (Stratagene): one min at 94° C., one min at each 2° C. increment from 44 to 66° C., and two min at 72° C. The amplicons were analyzed by agarose gel electrophoresis (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 7.20-7.43, Cold Spring Harbor, N.Y.), and the products were extracted from the gel, ligated into pT7Blue (Novagen) and transformed into E. coli XL1-Blue cells. Plasmid DNA was prepared from individual transformants and the inserts were fully sequenced (DyeDeoxy Terminator Cycle Sequencing, Applied Biosystems). In addition to previously described DNA fragments (Bohlmann, J., Steele, C. L., and Croteau, R. (1997) J. Biol. Chem. 272, 21784-21792), a new insert sequence was identified and was designated as probe 7 (SEQ ID NO: 63).
Library screening. For library screening, 200 ng of probe 7 (SEQ ID NO: 63) was amplified by PCR, and the resulting amplicon was gel purified, randomly labeled with [α-[0150] ³²P]dATP (Feinberg, A. P., and Vogelstein, B. (1984) Anal. Biochem. 137, 216-267) and used to screen replica filters of 5×10⁴plaques from the wound-induced grand fir stem cDNA library plated on E. coli LE392. Hybridization was performed for 20 h at 55° C. in 3×SSPE and 0.1% SDS. Filters were washed three times for 10 min at 55° C. in 3×SSPE with 0.1% SDS and exposed for 17 h to Kodak XAR film at −70° C. (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 7.20-7.43, Cold Spring Harbor, N.Y.). Forty λZAPII clones yielding positive signals with probe 7 (SEQ ID NO: 63) were purified through a second round of hybridization. Purified λZAPII clones were excised in vivo as Bluescript II SK⁻ phagemids and transformed into E. coli XLOLR according to the manufacturer's instructions (Stratagene). The size of each cDNA insert was determined by PCR using T3 (SEQ ID NO: 29) and T7 (SEQ ID NO: 30) promoter primers, and size-selected inserts (>1.5 kb) were partially sequenced from both ends to reveal several acquisitions of four unique clones, including apparent full-length versions of three (designated 7.30 (ag6) (SEQ ID NO: 64), 7.36 (ag8) (SEQ ID NO: 66) and 7.32 (ag11) (SEQ ID NO: 68)), and a fourth (7.31) (SEQ ID NO: 70) that was clearly 5′-truncated.
Rapid amplification of cDNA ends. To acquire the 5′-terminus corresponding to truncated cDNA clone ag7.31 (SEQ ID NO: 70), 5′-rapid amplification of cDNA ends (RACE) was carried out using the Marathon cDNA amplification system (Clontech) and the manufacturer's protocol. Nested reverse RACE primers specific for ag7.31 (SEQ ID NO: 70), designated 10-2 (5′-ACG AAG CTT CTT CTC CAC GG-3′)(SEQ ID NO: 71) and 10-4 (5′-GGA TCC CAT CTC TTA ACT GC-3′)(SEQ ID NO: 72), were used in combination with primer API (SEQ ID NO: 73) and AP2 (SEQ ID NO: 74) (Clontech), respectively. The resulting amplicon was cloned into vector pT7Blue (Novagen) and completely sequenced on both strands. The full length cDNA corresponding to clone ag7.31 (SEQ ID NO: 70) was then amplified by PCR using primers AG9F (5′-ATG GCT CTT GTT TCT ATC TTG CCC-3′) (SEQ ID NO: 75) and AG9R (5′-TTA CAA AGG CAC AGA CTC AAG GAC-3′)(SEQ ID NO: 76), ligated into pT7Blue and the resulting plasmid was designated pAG9 (SEQ ID NO: 77). [0151]
The foregoing similarity based cloning approach was designed to employ PCR primers E (SEQ ID NO: 21) and G (SEQ ID NO: 23) to generate new terpenoid synthase cDNA fragments for isolation and deployment as hybridization probes in screening an enriched library. By using wound-induced grand fir stem library cDNA as template, the combination of primers E (SEQ ID NO: 21) and G (SEQ ID NO: 23) amplified primarily a 1022 bp PCR product identical to probe 3 (SEQ ID NO: 24) and an additional, very minor product of 296 bp. This very low abundance 296 bp product was also amplified when only primer E (SEQ ID NO: 21) was used in the PCR reaction with the Grand fir cDNA library template in the absence of a second primer. Cloning and sequencing of this PCR product (designated probe 7) (SEQ ID NO: 63) unexpectedly revealed significant deduced sequence similarity with previously cloned grand fir terpene synthases in the region corresponding to amino acid Val[0152] ⁴²⁴-Leu⁵²¹of (−)-pinene synthase (SEQ ID NO: 4). The highest levels of identity were observed at the nucleic acid level with monoterpene synthase clones ag2.2 (SEQ ID NO: 1) (myrcene synthase, 74%), ag3.18 (SEQ ID NO: 3) (−)-pinene synthase, 69%) and ag10 (SEQ ID NO: 5) (−)-limonene synthase, 83%). Lower levels of similarity were noted with the sesquiterpene synthases agl (50%), ag4 (54%) and ag5 (57%), and still less with the diterpene synthase abietadiene synthase (46%), suggesting that this PCR product represented a fragment of a new monoterpene synthase. In retrospect, generation of this fragment can be explained based on two unexpected annealing events of primer E (SEQ ID NO: 21) to a monoterpene synthase cDNA in the regions corresponding to amino acids Val⁴²⁴-Ala⁴³²and Leu⁵¹³-Leu⁵²¹of (−)-pinene synthase (SEQ ID NO: 4). These binding sites for primer E (SEQ ID NO: 21) are significantly different from the amino acid sequence GE(K/T)(V/I)M6/D)EA (SEQ ID NO: 26) to which the degenerate primer was designed. With nucleic acid sequence identities between primer E (SEQ ID NO: 21) (23 nucleotides) and the corresponding two sites of recognition in previously acquired grand fir monoterpene synthases in the range of 44-57% and 48-56%, respectively, amplification of probe 7 (SEQ ID NO: 63) would not be predicted. Probe 7 (SEQ ID NO: 63) was subsequently employed for filter hybridization of the wound induced grand fir phage cDNA library.
Screening of 10[0153] ⁵cDNA phage plaques from the wounded grand fir stem library with probe 7 (SEQ ID NO: 63) yielded 40 positives of which 37 were isolated by one additional round of filter hybridization, excised in vivo and partially sequenced from both ends. Sequence analysis revealed four new, unique cDNA fragments represented by phage clones 7.30 (SEQ ID NO: 64), 7.36 (SEQ ID NO: 66) 7.31 (SEQ ID NO: 70) and 7.32 (SEQ ID NO: 68), of which clone 7.32 (SEQ ID NO: 68) was identical to probe 7 (SEQ ID NO: 63). Complete sequencing of these inserts and sequence comparison placed these genes into the gymnosperm Tpsd subfamily of plant terpenoid synthases (Bohlmann, J., Meyer-Gauen, G., and Croteau. R. (1998) Proc. Natl. Acad. Sci. USA 95, 4126-4133) with closest relationship to previously acquired grand fir monoterpene synthases. Alignment of the deduced amino acid sequences of the four new, presumptive terpene synthase fragments with extant monoterpene synthases indicated that clones 7.30 (SEQ ID NO: 64), 7.36 (SEQ ID NO: 66) and 7.32 (SEQ ID NO: 68) represented full-length versions, whereas clone 7.31 (SEQ ID NO: 70) was truncated at the 5′-terminus. A full-length clone corresponding to truncated cDNA 7.31 (SEQ ID NO: 70) was obtained by a 5′-RACE method. These full-length clones were designated ag6 (7.30) (SEQ ID NO: 64), ag8 (7.36) (SEQ ID NO: 66), ag9 (731) (SEQ ID NO: 77) and ag11 (7.32) (SEQ ID NO: 68), consistent with the nomenclature for other terpenoid synthases from grand fir (Bohlmann, J., Meyer-Gauen, G., and Croteau. R. (1998) Proc. Natl. Acad. Sci. USA 95, 4126-4133).
Sequence analysis. Inserts of all recombinant Bluescript plasmids and pSBET plasmids were completely sequenced on both strands via primer walking using the DyeDeoxy Terminator Cycle Sequencing method (Applied Biosystems). Sequence analysis was conducted using programs from the Genetics Computer Group (Genetics Computer Group (1996) Program Manual for the Wisconsin Package, Version 9.0, Genetics Computer Group, Madison, Wis.). [0154]
The sequences of clone ag6 (2013 bp with ORF of 1854 nt encoding 618 amino acids) (SEQ ID NO: 64), clone ag8(2186 bp with ORF of 1890 nt encoding 630 amino acids) (SEQ ID NO: 66), clone ag9 (1893 bp with ORF of 1890 nt encoding 630 amino acids) (SEQ ID NO: 77) and clone ag11 (2429 bp with ORF of 1911 nt encoding 637 amino acids) (SEQ ID NO: 68) revealed, in addition to overall similarities, other features characteristic of monoterpene synthases. The lengths of the deduced proteins (618-637 amino acids) and the predicted molecular weights (71,000-73,000) are in the range of other monoterpene synthases. The deduced proteins are larger than the sesquiterpene synthases, δ-selinene synthase (581 amino acids) and γ-humulene synthases (593 amino acids) (Steele, C. L., Crock, J., Bohlmann, J., and Croteau, R. (1998) [0155] J. Biol. Chem. 273, 2078-2089) but smaller than abietadiene synthase (868 amino acids) (Stofer Vogel, B., Wildung, M., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271, 23262-23268) and (E)-α-bisabolene synthase (817 amino acids) (Bohlmann, J., Crock, J., Jetter, R., and Croteau, R. (1998) Proc. Natl. Acad. Sci. USA 95, 6756-6761) from grand fir. As with other monoterpene synthases (Bohlmann, J., Meyer-Gauen, G., and Croteau. R. (1998) Proc. Natl. Acad. Sci. USA 95, 4126-4133), ag6 (SEQ ID NO: 64), ag8 (SEQ ID NO: 66), ag9 (SEQ ID NO: 77) and ag11 (SEQ ID NO: 68) appear to encode preproteins bearing an amino-terminal transit peptide for plastidial import of these nuclear gene products (Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (1998) Biochemisiry 37, 12213-12220; Turner, G., Gershenzon, J., Nielson, E. E., Froehlich, J. E., and Croteau, R. (1999) Plant Physiol., in press). In all cases, the amino-terminal 50-60 residues of the deduced sequences are rich in serine (15-21%) but have few acidic residues, consistent with such targeting peptides (Keegstra, K., Olsen, J. J., and Theg, S. M. (1989) Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 471-501; von Heijne, G., Stepphuhn, J., and Herrmann, R. G. (1989) Eur. J. Biochem. 180, 535-545). A tandem arginine element has recently been demonstrated to approximate the putative amino-terminus of mature monoterpene synthases, and to be involved in the initial diphosphate migration step of the coupled monoterpene cyclization reaction sequence in which geranyl diphosphate is isomerized to enzyme-bound linalyl diphosphate (Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (1998) Biochemistry 37, 12213-12220). The position of the tandem arginines is similar in ag6 (SEQ ID NO: 65) (R⁵⁹R⁶⁰), ag8 (SEQ ID NO: 67) (R⁶²R⁶³), ag9 (SEQ ID NO: 78) (R⁶⁵R⁶⁶) and ag11 (SEQ ID NO: 69) (R⁷¹R⁷²), and is conserved in all monoterpene synthases of angiosperm and gymnosperm origin in a position nine amino acids upstream of an absolutely conserved tryptophan residue (Bohlmann, J., Meyer-Gauen, G., and Croteau. R. (1998) Proc. Natl. Acad. Sci. USA 95, 4126-4133). All other previously described motifs of plant terpene synthases of the tpsa tpsb and tpsd subfamilies (Bohlmann, J., Meyer-Gauen, G., and Croteau. R. (1998) Proc. Natl. Acad. Sci. USA 95, 4126-4133), including the aspartate-rich DDXXD (SEQ ID NO: 45) element involved in coordinating the divalent metal ion for substrate binding (Ashby, M. N., and Edwards, P. A. (1990) J. Biol. Chem. 265, 13157-13164; Marrero, P. F., Poulter, C. D., and Edwards, P. A. (1992) J. Biol. Chem. 267, 21873-21878; Tarshis, L. C., Yan, M., Poulter, C. D., and Sacchettini, J. C. (1994) Biochemistry 33, 10871-10877; Cane, D. E., Sohng, J. K., Lamberson, C. R., Rudnicki, S. M., Wu, Z., Lloyd, M. D., Oliver, J. S., and Hubbard, B. R. (1994) Biochemistry 33, 5846-5857), are also found in the enzymes encoded by ag6 (SEQ ID NO: 65), ag8 (SEQ ID NO: 70), ag9 (SEQ ID NO: 78) and ag11 (SEQ ID NO: 69).
cDNA expression in [0156] E. coli and enzyme assays. The fully sequenced insert fragments of plasmids pAG6 (SEQ ID NO: 64), pAG8 (SEQ ID NO: 66), pAG9 (SEQ ID NO: 77) and pAG11 (SEQ ID NO: 68) were subcloned in-frame into the expression vector pSBETa (Schenk, P. M., Baumann, S., Mattes, R., and Steinbiss, H. (1995) BioTechniques 19, 196-200). First, internal NdeI sites and internal BamHI sites in cDNA clones pAG6 (SEQ ID NO: 64), pAG9 (SEQ ID NO: 77) and pAG11 (SEQ ID NO: 68) were eliminated by site directed mutagenesis using the Quick Change Mutagenesis system (Stratagene). Mutagenesis primers designated 6eBamHIF (5′-CAA TTA AGA GAT GGG ACC CGT CCG CGA TGG-3′) (SEQ ID NO: 79) and 6eBamHIR (5′-CCA TCG CGG ACG GGT CCC ATC TCT TAA TTG-3′) (SEQ ID NO: 80) were used to eliminate an internal BamHI site in pAG6 (SEQ ID NO: 64), 9eBamHIF (5′-GCA TTT AAG AGA TGG GAC CCG TCT GCC ACA G-3′) (SEQ ID NO: 81) and 9eBamHIR (5′-CTG TGG CAG ACG GGT CCC ATC TCT TAA ATG C-3′) (SEQ ID NO: 82) to eliminate an internal BamHI site in pAG9 (SEQ ID NO: 77), and 732eNdeIF (5′-CGA GAT GCC ATA CGT GAA TAC GCA G-3′) (SEQ ID NO: 83) and 732eNdeIR (5′-CTG CGT ATT CAC GTA TGG CAT CTC G-3′) (SEQ ID NO: 84) to eliminate an internal NdeI site in pAG11 (SEQ ID NO: 68).
To introduce suitable restriction sites for subcloning full-length versions and versions truncated to remove the presumptive plastidial targeting peptide of these enzymes (Bohlmann, J., Meyer-Gauen, G., and Croteau. R. (1998) [0157] Proc. Natl. Acad. Sci. USA 95, 4126-4133; Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (1998) Biochemistry 37, 12213-12220), fragments were amplified by PCR using primer 6-NdeI-M (5′-CTG ATA GCA AGC TCA TAT GGC TCT TCT TTC-3′) (SEQ ID NO: 85) or primer 6-NdeI-R (5′-GCC CAC GCG TCT CAT ATG AGA ATC AGT AGA TGC G-3′) (SEQ ID NO: 86) individually in combination with primer 6-BamHI (5′-CAC CCA TAG GGG ATC CTC AGT TAA TAT TG-3′) (SEQ ID NO: 87) for pAG6 (SEQ ID NO: 64), primer 8-NdeI-M (5′-TAA GCG AGC ACA TAT GGC TCT GGT TTC TTC-3′) (SEQ ID NO: 88) in combination with primer 8-BamHI (5′-GCA TAA ACG CAT AGC GGA TCC TAC ACC AA-3′) (SEQ ID NO: 89) for pAG8 (SEQ ID NO: 66), primer 9-NdeI-M (5′-CCC GGG GAT CGG ACA TAT GGC TCT TGT TTC-3′) (SEQ ID NO: 90) in combination with primer 9-BamHI (5′-GGT CGA CTC TAG AGG ATC CAC TAG TGA TAT GGA T-3′) (SEQ ID NO: 91) for pAG9 (SEQ ID NO: 77), and primer 11-NdeI-M (5′-GAA CAT ATG GCT CTC CTT TCT ATC GTA-3′) (SEQ ID NO: 92) or primer 11-NdeI-R (5′-GGT GGT GGT GTA CAT ATG AGA CGC ATA CGG G-3′) (SEQ ID NO: 93) in combination with primer 11-BamHI (5′-GAG ACT AGA CTG GAT CCC ATA TAC ACT GTA ATG G-3′) (SEQ ID NO: 94) for pAG11 (SEQ ID NO: 68). PCR reactions were performed in volumes of 50 μl containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1% Triton X-100, 5 μg bovine serum albumin, 200 μM of each dNTP, 0.1 μM of each primer, 2.5 units of recombinant Pfu DNA polymerase (Stratagene) and 100 ng plasmid DNA using the following program: denaturation at 94° C. for 1 min, annealing at 60° C. for 1 min, extension at 72° C. for 3.5 min; 35 cycles with final extension at 72° C. for 5 min. The resulting PCR products were purified by agarose gel electrophoresis and employed as templates for a second PCR amplification under the identical conditions but in a total volume of 250 μl each. Products from this secondary amplification were digested with the above indicated restriction enzymes, purified by ultrafiltration, and then ligated into NdeI/BamHI-digested pSBETa to yield the respective plasmids pSAG6(M), pSAG6(R), pSAG8(M), pSAG9(M), pSAG11(M) and pSAG11(R).
For use as controls in the expression of full-length and truncated forms, inserts of the previously described (Bohlmann, J., Steele, C. L., and Croteau, R. (1997) [0158] J. Biol. Chem. 272, 21784-21792) plasmids pGAG2.2 (myrcene synthase) (SEQ ID NO: 1) and pGAG3.18 ((−)-pinene synthase) (SEQ ID NO: 3) were prepared for subcloning into pSBETa by PCR amplification using primer 2-NdeI-M (5′-CAA AGG GAG CAC ATA TGG CTC TGG-3′) (SEQ ID NO: 95) or primer 2-NdeI-R (5′-CTG ATG ATG GTC ATA TGA GAC GCA TAG GTG-3′) (SEQ ID NO: 96) in combination with primer 2-BamHI (5′-GAC CTT ATT ATT ATG GAT CCG GTT ATA G-3′) (SEQ ID NO: 97) for pGAG2.2 (SEQ ID NO: 1), and primer 3-NdeI-R (5′-CCG ATG ATG GTC ATA TGA GAC GCA TGG GCG-3′) (SEQ ID NO: 98) in combination with primer 3-BamHI (5′-GGG CAT AGA TTT GAG CGG ATC CTA CAA AGG-3′) (SEQ ID NO: 99) for pGAG3.18 (SEQ ID NO: 3). Prior to insert subcloning, two internal BamHI sites and two internal NdeI sites were eliminated from the insert of pGAG3.18 (SEQ ID NO3:) by site directed mutagenesis using primers 3e1BamHIF (5′-CGT TTG GGA ATC CAT AGA CAT TTC-3′) (SEQ ID NO: 100), 3e1BamHIR (5′-GAA ATG TCT ATG GAT TCC CAA ACG-3′) (SEQ ID NO: 101), 3e2BamHIF (5′-GAA GAG ATG GGA CCC GTC CTC GAT AG-3′) (SEQ ID NO: 102), 3e2BamHIR (5′-CTA TCG AGG ACG GGT CCC ATC TCT TC-3′) (SEQ ID NO: 103), 3e1NdeIF (5′-GAA CAC GAA GTC CTA TGT GAA GAG C-3′) (SEQ ID NO: 104), 3e1NdeIR (5′-GCT CTT CAC ATA GGA CTT CGT GTT C-3′) (SEQ ID NO: 105), 3e3NdeIF (5′-GAT ACG CTC ACT TAT GCT CGG GAA G-3′) (SEQ ID NO: 106) and 3e2NdeIR (5′-CTT CCC GAG CAT AAG TGA GCG TAT C-3′) (SEQ ID NO: 107). Subcloning into pSBETa yielded pSAG2(M) and pSAG2(R) for myrcene synthase, and pSAG3(R) for (−)-pinene synthase. All recombinant pSBETa plasmids were confirmed by sequencing to insure that no errors had been introduced by the polymerase reactions, and were then transformed into E. coli BL21(DE3) by standard methods.
For functional expression, bacterial strains [0159] E. coli BL21(DE3)/pSAG2M, E. coli BL21(DE3)/pSAG2(R), E. coli BL21(DE3)/pSAG3(R), E. coli BL21(DE3)/pSAG6(M), E. coli BL21(DE3/pSAG6(R), E. coli BL21(DE3)/pSAG8(M), E. coli BL21(DE3)/pSAG9(M), E. coli BL21(DE3)/pSAG11(M) and E. coli BL21(DE3)/SAG11(R) were grown to A₆₀₀=0.5 at 37° C. in 5 ml of Luria-Bertani medium (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 7.20-7.43, Cold Spring Harbor, N.Y.) supplemented with 30 μg kanamycin/ml. Cultures were then induced by addition of 1 mM isopropyl-1-thio-β-D-galactopyranoside and grown for another 12 h at 20° C. Cells were harvested by centrifugation and disrupted by sonication followed by centrifugation, and the resulting soluble enzyme preparations were assayed for monoterpene, sesquiterpene and diterpene synthase activity using the appropriate radiolabeled substrate as described previously (Bohlmann, J., Steele, C. L., and Croteau, R. (1997) J. Biol. Chem. 272, 21784-21792). This assay involves the isolation and separation of terpene olefins and oxygenated terpenes by column chromatography on silica, followed by scintillation counting to determine conversion rate. GC-MS analysis is employed for product identification, by comparison of retention times and mass spectra to those of authentic standards (Bohlmann, J., Steele, C. L., and Croteau, R. (1997) J. Biol. Chem. 272, 21784-21792); assignment of absolute configuration is based upon chiral column capillary GC and matching of retention time to that of the corresponding enantiomer (Lewinsohn, E., Savage, T. J., Gijzen, M., and Croteau, R. (1993) Phytochem. Anal. 4, 220-225). To insure production of sufficient material for identification, the standard assay (Bohlmann, J., Steele, C. L., and Croteau, R. (1997) J. Biol. Chem. 272, 21784-21792) was scaled up by a factor of 20.
For functional characterization, cDNAs ag6 (SEQ ID NO: 64), ag8 (SEQ ID NO: 66), ag9 (SEQ ID NO: 77) and ag11 (SEQ ID NO: 68) were subcloned into the bacterial expression vector pSBET (Schenk, P. M., Baumann, S., Mattes, R., and Steinbiss, H. (1995) [0160] BioTechniques 19, 196-200). This vector employs the T7 RNA polymerase promoter and contains the argU gene for expression in E. coli of the tRNA using the rare arginine codons AGA and AGG that are commonly found in plant genes. pSBET constructs have been employed previously for successful bacterial expression of terpene synthases from gymnosperms and angiosperms (Steele, C. L., Crock, J., Bohlmann, J., and Croteau, R. (1998) J. Biol. Chem. 273, 2078-2089; Bohlmann, J., Steele, C. L., and Croteau, R. (1997) J. Biol. Chem. 272, 21784-21792; Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (1998) Biochemistry 37, 12213-12220).
Initially, the four pSBET plasmids pSAG6(M), pSAG8(M), pSAG9(M) and pSAG11(M), containing the full-length cDNA inserts of ag6 (SEQ ID NO: 64), ag8 (SEQ ID NO: 66), ag9 (SEQ ID NO: 77) and ag11 (SEQ ID NO: 68), respectively, were expressed in [0161] E. coli BL21(DE3). Extracts of the induced, transformed bacterial cells were assayed for monoterpene, sesquiterpene and diterpene synthase activity using the corresponding labeled C₁₀, C₁₅and C₂₀prenyl diphosphate substrates (Bohlmann, J., Steele, C. L., and Croteau, R. (1997) J. Biol. Chem. 272, 21784-21792). Enzymatic production of terpene olefin(s) was observed only with E. coli strains BL21(DE3)/pSAG8M) and BL21(DE3)/pSAG9(M) using [1-³H]geranyl diphosphate as substrate. Since it is known that the relatively large amino-terminal transit peptide of plant terpene synthases can impair expression of the functional preprotein in E. coli and promote the formation of intractable inclusion bodies (Williams, D. C., McGarvey, D. J., Katahira, E. J., and Croteau, R. (1998) Biochemistry 37, 12213-12220), the “pseudomature” forms of ag6 (SEQ ID NO: 64) and ag11 (SEQ ID NO: 68) were prepared by truncation of the cDNAs to insert a starting methionine immediately upstream of their tandem arginine elements (R⁵⁹R⁶⁰for ag6 (SEQ ID NO: 64), R⁷¹R⁷²for ag11 (SEQ ID NO: 68)) for expression in E. coli BL21 (DE3)/pSAG6(R) and E. coli BL21(DE3)/pSAG11(R). Similar truncations of previously characterized ag2.2 (SEQ ID NO: 1) (myrcene synthase, R⁶⁴R⁶⁵) and ag3.18 (SEQ ID NO: 3) ((−)-pinene synthase, (SEQ ID NO: 5) R⁶⁴R⁶⁵) (Bohlmann, J., Steele, C. L., and Croteau, R. (1997) J. Biol. Chem. 272, 21784-21792) were prepared for expression from pSBET as controls for the possible alteration of product yield and distribution resulting from truncation. These three latter constructs, pSAG2(M), pSAG2(R) and pSAG3(R), afforded high level expression of activity in BL21(DE3) cells, and revealed the identical product patterns previously observed for the myrcene synthase and pinene synthase preprotein forms expressed from pBluescript and pGEX vectors (Bohlmann, J., Steele, C. L., and Croteau, R. (1997) J. Biol. Chem. 272, 21784-21792). Thus, truncation directly upstream of the arginine pair does not alter product formation of these recombinant monoterpene synthases. When ag6 (SEQ ID NO: 64) and ag11 (SEQ ID NO: 68) were expressed as their truncated forms pSAG6(R) and pSAG11(R) in E. coli BL21(DE3), and the extracts assayed for all three terpene synthase activities, only geranyl diphosphate yielded high levels of terpene olefin products.
Monoterpenes generated at preparative scale from geranyl diphosphate by the recombinant enzymes encoded by ag6 (SEQ ID NO: 64), ag8 (SEQ ID NO: 66), ag9 (SEQ ID NO: 77) and ag11 (SEQ ID NO: 68) were analyzed by GC-MS and chiral phase (β-cyclodextrin) capillary GC, and identified by comparison of retention times and mass spectra to authentic standards (FIGS. [0162] 6-9). The synthase encoded by ag6 (SEQ ID NO: 65) was shown to produce three principal products (FIG. 6A), the major one of which was identified as (−)-1S,4R-camphene (54%) (FIGS. 6B and C), followed by (−)-1S,5S-α-pinene (32%) and (−)-4S-limonene (7%) which were identified by similar means (data not shown (These additional data may be accessed at website www.wsu.edu/˜ibc/faculty/rc.html)). The enzyme encoded by ag6 (SEQ ID NO: 65) is therefore designated as (−)-1S,4R-camphene synthase. Interestingly, the (−)-camphene synthase (SEQ ID NO: 65) is not as stereoselective as other monoterpene synthases in producing both (−)-α-pinene and (+)-α-pinene as coproducts in a ratio of 95:5; the (−)-enantiomer dominates to the extent of more than 99% in this pinene isomer formed by (−)-pinene synthase from grand fir (Bohlmann, J., Steele, C. L., and Croteau, R. (1997) J. Biol. Chem. 272, 21784-21792). Soluble enzyme preparations from E. coli BL21(DE3)/pSAG8(M) converted geranyl diphosphate to four products (FIG. 7A) with β-phellandrene (52%) as the major olefin (FIGS. 7B and C), and lesser amounts of (−)-1S,5S-β-pinene (34%), (−)-1S,5S-α-pinene (8.5%) and (−)-4S-limonene (6%) identified by similar means (data not shown (these additional data may be accessed at website www.wsu.edu/˜ibc/faculty/rc.html)). The stereochemistry of β-phellandrene was not confirmed directly, since only the authentic (+)-enantiomer was available as a standard for chiral phase GC analysis. Nevertheless, stereochemical considerations based on the established absolute configuration of the co-products, and the natural occurrence of (−)-4S-β-phellandrene in the turpentine (Lewinsohn, E., Savage, T. J., Gijzen, M., and Croteau, R. (1993) Phytochem. Anal. 4, 220-225), suggest that the biosynthetic product is the mechanistically related (−)-4S-antipode derived via the (+)-3S-linalyl diphosphate intermediate (Croteau, R. (1987) Chem. Rev. 87, 929-954; Wise, M. L., and Croteau, R. (1998) in Comprehensive Natural Products Chemistry: Isoprenoids (Cane, D. E., Ed.), Vol. 2, pp. 97-153, Elsevier Science, Oxford). The product of the ag8 gene (SEQ ID NO: 67) is therefore designated (−)-4S-β-phellandrene synthase.
The enzyme encoded by cDNA clone ag9 (SEQ ID NO: 78) also produced several monoterpenes from geranyl diphosphate (FIG. 8), with the achiral olefin terpinolene identified as the major product (42%) (FIGS. 8B and C). In addition to minor amounts of four unidentified olefins (FIG. 8A), (−)-α-pinene (18%), (−)-limonene (11%) and (−)-β-pinene (10%) were also identified as significant biosynthetic products by mass spectrometric and chromatographic analysis (data not shown (These additional data may be accessed at website www.wsu.edu/˜ibc/faculty/rc.html)), indicating that the entire product set was derived in stereochemically consistent fashion via (+)-3S-linalyl diphosphate as intermediate (Croteau, R. (1987) [0163] Chem. Rev. 87, 929-954; Wise, M. L., and Croteau, R. (1998) in Comprehensive Natural Products Chemistry: Isoprenoids (Cane, D. E., Ed.), Vol. 2, pp. 97-153, Elsevier Science, Oxford). The enzyme encoded by ag9 (SEQ ID NO: 78) is designated terpinolene synthase based on the principal olefinic product. cDNA clone ag11 (SEQ ID NO: 68) also encodes a multiple product monoterpene synthase FIG. 9) with the two most abundant products identified as (−)-4S-limonene (35%) and (−)-1S,5S-α-pinene (24%) (FIGS. 9B and C), with lesser amounts of β-phellandrene (20%, presumably also the (−)-antipode), (−)-1S,5S-β-pinene (11%), and (−)-1S,5S-sabinene (10%) (data not shown (These additional data may be accessed at website www.wsu.edu/˜ibc/faculty/rc.html)). This is the first recombinant monoterpene synthase to produce such a range of cyclic olefins, none of which truly dominate the profile (FIG. 9A). Based on the close sequence relatedness between clone ag11 (SEQ ID NO: 68) and the previously described (−)-limonene synthase of grand fir (ag10 (SEQ ID NO: 5), which produces about 80% (−)-limonene (Bohlmann, J., Steele, C. L., and Croteau, R. (1997) J. Biol. Chem. 272, 21784-21792)), ag11 (SEQ ID NO: 69) was previously considered to be a likely (−)-limonene synthase. However, given the significant production of (−)-α-pinene by this cyclase, unlike the ag10 (−)-limonene synthase (SEQ ID NO: 6), it seems appropriate to designate the product of the ag11 gene (SEQ ID NO: 69) as (−)-limonene/(−)-α-pinene synthase to clearly distinguish the two. The (−)-limonene/(−)-α-pinene synthase (ag11) (SEQ ID NO: 69) and the (−)-limonene synthase (ag10) (SEQ ID NO: 6) share 93% similarity and 91% identity at the deduced amino acid level, demonstrating that less than 10% sequence divergence is sufficient to result in a significantly different product outcome. Interestingly, all sequence differences between ag11 (SEQ ID NO: 69) and ag10 (SEQ ID NO: 6) are confined to the carboxy-terminal half of these proteins, which is thought to comprise the active site region involved in the cyclization step(s) catalyzed by sesquiterpene synthases and presumably other terpene synthases as well (Starks, C. M., Back, K., Chappell, J., and Noel, J. P. (1997) Science 277, 1815-1820). This finding is relevant to the rational redesign of terpene synthases, and to the evolution of these catalysts which almost certainly involves gene duplication and modification as the basis for generating new terpenoid synthase function (Bohlmann, J., Meyer-Gauen, G., and Croteau. R. (1998) Proc. Natl. Acad. Sci. USA 95, 4126-4133).
cDNAs encoding (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase and (−)-limonene/(−)-α-pinene synthase have not been described previously from any source. Together with the cloned myrcene synthase (SEQ ID NO: 1), (−)-limonene synthase (SEQ ID NO: 5) and (−)-pinene synthase (SEQ ID NO: 3), these seven enzymes account for the production of most, but not all, monoterpenes of the constitutive turpentine (Lewinsohn, E., Savage, T. J., Gijzen, M., and Croteau, R. (1993) [0164] Phytochem. Anal. 4, 220-225) and wound-induced resin (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289, 267-273) of grand fir. The cDNA encoding a constitutive and/or inducible (+)-β-carene synthase (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289, 267-273; Savage, T. J., and Croteau, R. (1993) Arch. Biochem. Biophys. 305, 581-587) has thus far eluded detection. Based on analytical results (Lewinsohn, E., Savage, T. J., Gijzen, M., and Croteau, R. (1993) Phytochem. Anal. 4, 220-225) and in vitro assays (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289, 267-273), the β-phellandrene synthase (SEQ ID NO: 67) contributes principally to production of the constitutive turpentine. The terpinolene synthase (SEQ ID NO: 78) may also represent a primarily constitutive enzyme; however, the complex and overlapping product profiles of these synthases do not allow unambiguous assignment of their functional role(s) in constitutive resin synthesis or the induced response without more detailed RNA blot analysis. A recent analytical survey of a small grand fir population for different chemotypes in both constitutive resin production and the induced response indicates considerable variation in the constitutive and inducible deployment of the members of this tpsd multiple gene family (Katoh, S., and Croteau, R. (1998) Phytochemistry 47, 577-582).

EXAMPLE 12

Hybridization Conditions [0165]
Presently preferred nucleic acid molecules of the present invention hybridize under stringent conditions to either one or both of hybridization probe A and hybridization probe B (or to their complementary antisense sequences). Hybridization probe A has the nucleic acid sequence of the portion of SEQ ID NO: 3 extending from nucleotide 1560 to nucleotide 1694. Hybridization probe B has the nucleic acid sequence of the portion of SEQ ID NO: 5 extending from nucleotide 1180 to nucleotide 1302. High stringency conditions are defined as hybridization in 5×SSC at 65° C. for 16 hours, followed by two washes in 2×SSC at 20° C. to 26° C. for fifteen minutes per wash, followed by two washes in 0.2×SSC at 65° C. for twenty minutes per wash. Moderate stringency conditions are defined as hybridization in 3×SSC at 65° C. for 16 hours, followed by two washes in 2×SSC at 20° C. to 26° C. for twenty minutes per wash, followed by one wash in 0.5×SSC at 55° C. for thirty minutes. Low stringency conditions are defined as hybridization in 3×SSC at 65° C. for 16 hours, followed by two washes in 2×SSC at 20° C. to 26° C. for twenty minutes per wash. [0166]

EXAMPLE 13

Characteristics of Presently Preferred Monoterpene Synthase Proteins of the Invention [0167]
Presently preferred monoterpene synthase proteins of the present invention have the following characteristics: a preprotein molecular weight of from about 71 kiloDaltons (kDa) to about 73 kDa; a mature protein (excluding plastidial transit peptide) molecular weight of from about 60 kDa to about 67 kDa; isoelectric point (pI) of from about 4.5 to about 5.0 as determined by isoelectric focussing; a mature protein pH optimum of from about 7.5 to about 8.0; utilization of geranyl diphosphate as a substrate with K[0168] _mless than 15 μM; a requirement for a divalent metal ion as a cofactor, with manganese (Mn²⁺) being preferred over magnesium (Mg²⁺), the K_mfor binding of Mn²⁺ being less than 50 μM; biological activity is enhanced by the presence of K⁺ ions at a concentration of 100 μM; the presence of the sequence motif DDXXD (SEQ ID NO: 45) within the carboxy terminal half of the protein, and the presence of two tandem arginines immediately following the C-terminal end of the plastidial transit peptide. The foregoing characteristics apply to the mature protein, unless otherwise stated.

EXAMPLE 14

Alteration of Monoterpene Levels and Composition in Plant Seeds [0169]
In accordance with the present invention, methods for increasing production of monoterpene compounds in a plant, particularly in plant seeds, are provided. The methods involve transforming a plant cell with a nucleic acid sequence encoding at least one monoterpene synthase, such as those encoded by the nucleic acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68 and SEQ ID NO: 77. This has the effect of altering monoterpene biosynthesis, thereby increasing the production of monoterpenes, as well as providing novel seed oils having desirable monoterpene compositions. In this manner, the transformed seed provides a factory for the production of modified oils. The modified oil itself may be used and/or the compounds in the oils can be isolated. Thus, the present invention allows for the production of particular monoterpenes of interest as well as specialty oils. [0170]
The nucleic acid encoding the monoterpene synthases of the present invention can be used in expression cassettes for expression in the transformed plant tissues. To alter the monoterpene levels in a plant of interest, the plant is transformed with at least one expression cassette comprising a transcriptional initiation region linked to a nucleic acid sequence encoding a monoterpene synthase. Such an expression cassette is provided with a plurality of restriction sites for insertion of the nucleic acid sequence encoding a monoterpene synthase so that it is under the transcriptional regulation of the regulatory regions. [0171]
The transcriptional initiation sequence may be native or analogous to the host or foreign or heterologous to the host. In this regard, the term “foreign” means that the transcriptional initiation sequence is not found in the wild-type host into which the transcriptional initiation region is introduced. [0172]
Of particular interest are those transcriptional initiation regions associated with storage proteins, such as napin, cruciferin, β-conglycinin, phaseolin, globulin or the like, and proteins involved in fatty acid biosynthesis, such as acyl carrier protein (ACP). See, U.S. Pat. No. 5,420,034, herein incorporated by reference. [0173]
The transcriptional cassette will preferably include, in the 5′ to 3′ direction of transcription, a transcriptional and translational initiation region, a monoterpene synthase DNA sequence of interest, and a transcriptional and translational termination region functional in plants. The termination region may be from the same organism as the transcriptional initiation region, may be from the same organism as the monoterpene synthase DNA, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of [0174] A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. Other termination sequences are set forth in Guerineau et al., (1991), Mol. Gen. Genet., 262:141-144; Proudfoot, (1991), Cell, 64:671-674; Sanfacon et al., (1991), Genes Dev., 5:141-149; Mogen et al., (1990), Plant Cell, 2:1261-1272; Munroe et al., (1990), Gene, 91:151-15 8; Ballas et al., (1989), Nucleic Acids Res., 17:7891-7903; Joshi et al., (1987), Nucleic Acid Res., 15:9627-9639).
In the presently preferred form of the invention, a nucleic acid sequence encoding a monoterpene synthase protein will be targeted to plastids, such as chloroplasts, for expression. Thus, the nucleic acid sequence, or sequences, encoding a monoterpene synthase protein, or proteins, may be inserted into the plastid for expression with appropriate plastid constructs and regulatory elements. Alternatively, nuclear transformation may be used in which case the expression cassette will contain a nucleic acid sequence encoding a transit peptide to direct the monoterpene biosynthesis enzyme of interest to the plastid. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) [0175] Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and, Shah et al. (1986) Science 233:478-481. Nucleic acid sequences encoding monoterpene synthases of the present invention may utilize native or heterologous transit peptides.
The construct may also include any other necessary regulators such as plant translational consensus sequences (Joshi, C. P., (1987), [0176] Nucleic Acids Research, 15:6643-6653), introns (Luehrsen and Walbot, (1991), Mol. Gen. Genet., 225:81-93) and the like, operably linked to a nucleotide sequence encoding a monoterpene synthase of the present invention.
It may be beneficial to include 5′ leader sequences in the expression cassette which can act to enhance translation. Translation leaders are known in the art and include: picomavirus leaders, for example, EMCV leader ([0177] Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. (1989) PNAS USA 86:6126-6130); polyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology, 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D. G., and Sarnow, P. (1991), Nature, 353:90-94; untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L. (1987), Nature, 325:622-625; tobacco mosaic virus leader (TMV), (Gallie, D. R. et al. (1989), Molecular Biology of RNA, pages 237-256; and maize chlorotic mottle virus leader (MCMV) (Lommel, S. A. et al. (1991), Virology, 81:382-385. See also, Della-Cioppa et al., (1987), Plant Physiology, 84:965-968.
Depending upon where the monoterpene synthase sequence of interest is to be expressed, it may be desirable to synthesize the sequence with plant preferred codons, or alternatively with chloroplast preferred codons. The plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in the largest amount in the particular plant species of interest. See, EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991) [0178] Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Research 17:477-498. In this manner, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the nucleic acid sequence encoding a monoterpene synthase protein may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used. For the construction of chloroplast preferred genes, see U.S. Pat. No. 5,545,817.
In preparing the transcription cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and in the proper reading frame. Towards this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, or the like may be employed, where insertions, deletions or substitutions, such as transitions and transversions, may be involved. [0179]
The recombinant DNA molecules of the invention can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant, i.e., monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al. (1986) [0180] BioTechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium mediated transformation (Hinchee et al. (1988) Biotechnology 6:915-921) and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; and McCabe et al. (1988) Biotechnology 6:923-926). Also see, Weissinger et al. (1988) Annual Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA, 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fronun et al. (1990) Biotechnology 8:833-839; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618 (maize).
Alternatively, a plant plastid can be transformed directly. Stable transformation of chloroplasts has been reported in higher plants, see, for example, SVAB et al. (1990) [0181] Proc. Nat'l. Acad. Sci. USA 87:85268530; SVAB & Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Staub & Maliga (1993) Embo J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. In such methods, plastid gene expression can be accomplished by use of a plastid gene promoter or by trans-activation of a silent plastid-borne transgene positioned for expression from a selective promoter sequence such as that recognized by T7 RNA polymerase. The silent plastid gene is activated by expression of the specific RNA polymerase from a nuclear expression construct and targeting of the polymerase to the plastid by use of a transit peptide. Tissue-specific expression may be obtained in such a method by use of a nuclear-encoded and plastid-directed specific RNA polymerase expressed from a suitable plant tissue specific promoter. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
The cells which have been transformed may be grown into plants by a variety of art-recognized means. See, for example, McConnick et al., [0182] Plant Cell Reports (1986), 5:81-84. These plants may then be grown, and either selfed or crossed with a different plant strain, and the resulting homozygotes or hybrids having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.
As a host cell, any plant variety may be employed. Of particular interest, are plant species which provide seeds of commercial value. For the most part, plants will be chosen where the seed is produced in high amounts, a seed-specific product of interest is involved, or the seed or a seed part is edible. Seeds of interest in the practice of the present invention include, but are not limited to, the oil seeds, such as oilseed Brassica seeds, cotton seeds, soybean, safflower, sunflower, coconut, palm, and the like; grain seeds such as wheat, barley, oats, amaranth, flax, rye, triticale, rice and corn; other edible seeds or seeds with edible parts including pumpkin, squash, sesame, poppy, grape, mung beans, peanut, peas, beans, radish, alfalfa, cocoa, and coffee; and tree nuts such as walnuts, almonds, pecans, and chick-peas. [0183]
While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. [0184]

0

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 107

<210> SEQ ID NO 1

<211> LENGTH: 2196

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (69)..(1952)

<223> OTHER INFORMATION:

<400> SEQUENCE: 1

tgccggcacg aggttatctt gagcttcctc catataggcc aacacatatc atatcaaagg 60

gagcaaga atg gct ctg gtt tct atc tca ccg ttg gct tcg aaa tct tgc 110

Met Ala Leu Val Ser Ile Ser Pro Leu Ala Ser Lys Ser Cys

1 5 10

ctg cgc aag tcg ttg atc agt tca att cat gaa cat aag cct ccc tat 158

Leu Arg Lys Ser Leu Ile Ser Ser Ile His Glu His Lys Pro Pro Tyr

15 20 25 30

aga aca atc cca aat ctt gga atg cgt agg cga ggg aaa tct gtc acg 206

Arg Thr Ile Pro Asn Leu Gly Met Arg Arg Arg Gly Lys Ser Val Thr

35 40 45

cct tcc atg agc atc agt ttg gcc acc gct gca cct gat gat ggt gta 254

Pro Ser Met Ser Ile Ser Leu Ala Thr Ala Ala Pro Asp Asp Gly Val

50 55 60

caa aga cgc ata ggt gac tac cat tcc aat atc tgg gac gat gat ttc 302

Gln Arg Arg Ile Gly Asp Tyr His Ser Asn Ile Trp Asp Asp Asp Phe

65 70 75

ata cag tct cta tca acg cct tat ggg gaa ccc tct tac cag gaa cgt 350

Ile Gln Ser Leu Ser Thr Pro Tyr Gly Glu Pro Ser Tyr Gln Glu Arg

80 85 90

gct gag aga tta att gtg gag gta aag aag ata ttc aat tca atg tac 398

Ala Glu Arg Leu Ile Val Glu Val Lys Lys Ile Phe Asn Ser Met Tyr

95 100 105 110

ctg gat gat gga aga tta atg agt tcc ttt aat gat ctc atg caa cgc 446

Leu Asp Asp Gly Arg Leu Met Ser Ser Phe Asn Asp Leu Met Gln Arg

115 120 125

ctt tgg ata gtc gat agc gtt gaa cgt ttg ggg ata gct aga cat ttc 494

Leu Trp Ile Val Asp Ser Val Glu Arg Leu Gly Ile Ala Arg His Phe

130 135 140

aag aac gag ata aca tca gct ctg gat tat gtt ttc cgt tac tgg gag 542

Lys Asn Glu Ile Thr Ser Ala Leu Asp Tyr Val Phe Arg Tyr Trp Glu

145 150 155

gaa aac ggc att gga tgt ggg aga gac agt att gtt act gat ctc aac 590

Glu Asn Gly Ile Gly Cys Gly Arg Asp Ser Ile Val Thr Asp Leu Asn

160 165 170

tca act gcg ttg ggg ttt cga act ctt cga tta cac ggg tac act gta 638

Ser Thr Ala Leu Gly Phe Arg Thr Leu Arg Leu His Gly Tyr Thr Val

175 180 185 190

tct cca gag gtt tta aaa gct ttt caa gat caa aat gga cag ttt gta 686

Ser Pro Glu Val Leu Lys Ala Phe Gln Asp Gln Asn Gly Gln Phe Val

195 200 205

tgc tcc ccc ggt cag aca gag ggt gag atc aga agc gtt ctt aac tta 734

Cys Ser Pro Gly Gln Thr Glu Gly Glu Ile Arg Ser Val Leu Asn Leu

210 215 220

tat cgg gct tcc ctc att gcc ttc cct ggt gag aaa gtt atg gaa gaa 782

Tyr Arg Ala Ser Leu Ile Ala Phe Pro Gly Glu Lys Val Met Glu Glu

225 230 235

gct gaa atc ttc tcc aca aga tat ttg aaa gaa gct cta caa aag att 830

Ala Glu Ile Phe Ser Thr Arg Tyr Leu Lys Glu Ala Leu Gln Lys Ile

240 245 250

cca gtc tcc gct ctt tca caa gag ata aag ttt gtt atg gaa tat ggc 878

Pro Val Ser Ala Leu Ser Gln Glu Ile Lys Phe Val Met Glu Tyr Gly

255 260 265 270

tgg cac aca aat ttg cca aga ttg gaa gca aga aat tac ata gac aca 926

Trp His Thr Asn Leu Pro Arg Leu Glu Ala Arg Asn Tyr Ile Asp Thr

275 280 285

ctt gag aaa gac acc agt gca tgg ctc aat aaa aat gct ggg aag aag 974

Leu Glu Lys Asp Thr Ser Ala Trp Leu Asn Lys Asn Ala Gly Lys Lys

290 295 300

ctt tta gaa ctt gca aaa ttg gag ttc aat ata ttt aac tcc tta caa 1022

Leu Leu Glu Leu Ala Lys Leu Glu Phe Asn Ile Phe Asn Ser Leu Gln

305 310 315

caa aag gaa tta caa tat ctt ttg aga tgg tgg aaa gag tcg gat ttg 1070

Gln Lys Glu Leu Gln Tyr Leu Leu Arg Trp Trp Lys Glu Ser Asp Leu

320 325 330

cct aaa ttg aca ttt gct cgg cat cgt cat gtg gaa ttc tac act ttg 1118

Pro Lys Leu Thr Phe Ala Arg His Arg His Val Glu Phe Tyr Thr Leu

335 340 345 350

gcc tct tgt att gcc att gac cca aaa cat tct gca ttc aga cta ggc 1166

Ala Ser Cys Ile Ala Ile Asp Pro Lys His Ser Ala Phe Arg Leu Gly

355 360 365

ttc gcc aaa atg tgt cat ctt gtc aca gtt ttg gac gat att tac gac 1214

Phe Ala Lys Met Cys His Leu Val Thr Val Leu Asp Asp Ile Tyr Asp

370 375 380

act ttt gga acg att gac gag ctt gaa ctc ttc aca tct gca att aag 1262

Thr Phe Gly Thr Ile Asp Glu Leu Glu Leu Phe Thr Ser Ala Ile Lys

385 390 395

aga tgg aat tca tca gag ata gaa cac ctt cca gaa tat atg aaa tgt 1310

Arg Trp Asn Ser Ser Glu Ile Glu His Leu Pro Glu Tyr Met Lys Cys

400 405 410

gtg tac atg gtc gtg ttt gaa act gta aat gaa ctg aca cga gag gcg 1358

Val Tyr Met Val Val Phe Glu Thr Val Asn Glu Leu Thr Arg Glu Ala

415 420 425 430

gag aag act caa ggg aga aac act ctc aac tat gtt cga aag gct tgg 1406

Glu Lys Thr Gln Gly Arg Asn Thr Leu Asn Tyr Val Arg Lys Ala Trp

435 440 445

gag gct tat ttt gat tca tat atg gaa gaa gca aaa tgg atc tct aat 1454

Glu Ala Tyr Phe Asp Ser Tyr Met Glu Glu Ala Lys Trp Ile Ser Asn

450 455 460

ggt tat ctg cca atg ttt gaa gag tac cat gag aat ggg aaa gtg agc 1502

Gly Tyr Leu Pro Met Phe Glu Glu Tyr His Glu Asn Gly Lys Val Ser

465 470 475

tct gca tat cgc gta gca aca ttg caa ccc atc ctc act ttg aat gca 1550

Ser Ala Tyr Arg Val Ala Thr Leu Gln Pro Ile Leu Thr Leu Asn Ala

480 485 490

tgg ctt cct gat tac atc ttg aag gga att gat ttt cca tcc agg ttc 1598

Trp Leu Pro Asp Tyr Ile Leu Lys Gly Ile Asp Phe Pro Ser Arg Phe

495 500 505 510

aat gat ttg gca tcg tcc ttc ctt cgg cta cga ggt gac aca cgc tgc 1646

Asn Asp Leu Ala Ser Ser Phe Leu Arg Leu Arg Gly Asp Thr Arg Cys

515 520 525

tac aag gcc gat agg gat cgt ggt gaa gaa gct tcg tgt ata tca tgt 1694

Tyr Lys Ala Asp Arg Asp Arg Gly Glu Glu Ala Ser Cys Ile Ser Cys

530 535 540

tat atg aaa gac aat cct gga tca acc gaa gaa gat gcc ctc aat cat 1742

Tyr Met Lys Asp Asn Pro Gly Ser Thr Glu Glu Asp Ala Leu Asn His

545 550 555

atc aat gcc atg gtc aat gac ata atc aaa gaa tta aat tgg gaa ctt 1790

Ile Asn Ala Met Val Asn Asp Ile Ile Lys Glu Leu Asn Trp Glu Leu

560 565 570

cta aga tcc aac gac aat att cca atg ctg gcc aag aaa cat gct ttt 1838

Leu Arg Ser Asn Asp Asn Ile Pro Met Leu Ala Lys Lys His Ala Phe

575 580 585 590

gac ata aca aga gct ctc cac cat ctc tac ata tat cga gat ggc ttt 1886

Asp Ile Thr Arg Ala Leu His His Leu Tyr Ile Tyr Arg Asp Gly Phe

595 600 605

agt gtt gcc aac aag gaa aca aaa aaa ttg gtt atg gaa aca ctc ctt 1934

Ser Val Ala Asn Lys Glu Thr Lys Lys Leu Val Met Glu Thr Leu Leu

610 615 620

gaa tct atg ctt ttt taa ctataaccat atccataata ataagctcat 1982

Glu Ser Met Leu Phe

625

aatgctaaat tattggcctt atgacatagt ttatgtatgt acttgtgtga attcaatcat 2042

atcgtgtggg tatgattaaa aagctagagc ttactaggtt agtaacatgg tgataaaagt 2102

tataaaatgt gagttataga gatacccatg ttgaataatg aattacaaaa agagaaattt 2162

atgtagaata agattggaag cttttcaatt gttt 2196

<210> SEQ ID NO 2

<211> LENGTH: 627

<212> TYPE: PRT

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 2

Met Ala Leu Val Ser Ile Ser Pro Leu Ala Ser Lys Ser Cys Leu Arg

1 5 10 15

Lys Ser Leu Ile Ser Ser Ile His Glu His Lys Pro Pro Tyr Arg Thr

20 25 30

Ile Pro Asn Leu Gly Met Arg Arg Arg Gly Lys Ser Val Thr Pro Ser

35 40 45

Met Ser Ile Ser Leu Ala Thr Ala Ala Pro Asp Asp Gly Val Gln Arg

50 55 60

Arg Ile Gly Asp Tyr His Ser Asn Ile Trp Asp Asp Asp Phe Ile Gln

65 70 75 80

Ser Leu Ser Thr Pro Tyr Gly Glu Pro Ser Tyr Gln Glu Arg Ala Glu

85 90 95

Arg Leu Ile Val Glu Val Lys Lys Ile Phe Asn Ser Met Tyr Leu Asp

100 105 110

Asp Gly Arg Leu Met Ser Ser Phe Asn Asp Leu Met Gln Arg Leu Trp

115 120 125

Ile Val Asp Ser Val Glu Arg Leu Gly Ile Ala Arg His Phe Lys Asn

130 135 140

Glu Ile Thr Ser Ala Leu Asp Tyr Val Phe Arg Tyr Trp Glu Glu Asn

145 150 155 160

Gly Ile Gly Cys Gly Arg Asp Ser Ile Val Thr Asp Leu Asn Ser Thr

165 170 175

Ala Leu Gly Phe Arg Thr Leu Arg Leu His Gly Tyr Thr Val Ser Pro

180 185 190

Glu Val Leu Lys Ala Phe Gln Asp Gln Asn Gly Gln Phe Val Cys Ser

195 200 205

Pro Gly Gln Thr Glu Gly Glu Ile Arg Ser Val Leu Asn Leu Tyr Arg

210 215 220

Ala Ser Leu Ile Ala Phe Pro Gly Glu Lys Val Met Glu Glu Ala Glu

225 230 235 240

Ile Phe Ser Thr Arg Tyr Leu Lys Glu Ala Leu Gln Lys Ile Pro Val

245 250 255

Ser Ala Leu Ser Gln Glu Ile Lys Phe Val Met Glu Tyr Gly Trp His

260 265 270

Thr Asn Leu Pro Arg Leu Glu Ala Arg Asn Tyr Ile Asp Thr Leu Glu

275 280 285

Lys Asp Thr Ser Ala Trp Leu Asn Lys Asn Ala Gly Lys Lys Leu Leu

290 295 300

Glu Leu Ala Lys Leu Glu Phe Asn Ile Phe Asn Ser Leu Gln Gln Lys

305 310 315 320

Glu Leu Gln Tyr Leu Leu Arg Trp Trp Lys Glu Ser Asp Leu Pro Lys

325 330 335

Leu Thr Phe Ala Arg His Arg His Val Glu Phe Tyr Thr Leu Ala Ser

340 345 350

Cys Ile Ala Ile Asp Pro Lys His Ser Ala Phe Arg Leu Gly Phe Ala

355 360 365

Lys Met Cys His Leu Val Thr Val Leu Asp Asp Ile Tyr Asp Thr Phe

370 375 380

Gly Thr Ile Asp Glu Leu Glu Leu Phe Thr Ser Ala Ile Lys Arg Trp

385 390 395 400

Asn Ser Ser Glu Ile Glu His Leu Pro Glu Tyr Met Lys Cys Val Tyr

405 410 415

Met Val Val Phe Glu Thr Val Asn Glu Leu Thr Arg Glu Ala Glu Lys

420 425 430

Thr Gln Gly Arg Asn Thr Leu Asn Tyr Val Arg Lys Ala Trp Glu Ala

435 440 445

Tyr Phe Asp Ser Tyr Met Glu Glu Ala Lys Trp Ile Ser Asn Gly Tyr

450 455 460

Leu Pro Met Phe Glu Glu Tyr His Glu Asn Gly Lys Val Ser Ser Ala

465 470 475 480

Tyr Arg Val Ala Thr Leu Gln Pro Ile Leu Thr Leu Asn Ala Trp Leu

485 490 495

Pro Asp Tyr Ile Leu Lys Gly Ile Asp Phe Pro Ser Arg Phe Asn Asp

500 505 510

Leu Ala Ser Ser Phe Leu Arg Leu Arg Gly Asp Thr Arg Cys Tyr Lys

515 520 525

Ala Asp Arg Asp Arg Gly Glu Glu Ala Ser Cys Ile Ser Cys Tyr Met

530 535 540

Lys Asp Asn Pro Gly Ser Thr Glu Glu Asp Ala Leu Asn His Ile Asn

545 550 555 560

Ala Met Val Asn Asp Ile Ile Lys Glu Leu Asn Trp Glu Leu Leu Arg

565 570 575

Ser Asn Asp Asn Ile Pro Met Leu Ala Lys Lys His Ala Phe Asp Ile

580 585 590

Thr Arg Ala Leu His His Leu Tyr Ile Tyr Arg Asp Gly Phe Ser Val

595 600 605

Ala Asn Lys Glu Thr Lys Lys Leu Val Met Glu Thr Leu Leu Glu Ser

610 615 620

Met Leu Phe

625

<210> SEQ ID NO 3

<211> LENGTH: 2018

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (6)..(1892)

<223> OTHER INFORMATION:

<400> SEQUENCE: 3

cagca atg gct cta gtt tct acc gca ccg ttg gct tcc aaa tca tgc ctg 50

Met Ala Leu Val Ser Thr Ala Pro Leu Ala Ser Lys Ser Cys Leu

1 5 10 15

cac aaa tcg ttg atc agt tct acc cat gag ctt aag gct ctc tct aga 98

His Lys Ser Leu Ile Ser Ser Thr His Glu Leu Lys Ala Leu Ser Arg

20 25 30

aca att cca gct cta gga atg agt agg cga ggg aaa tct atc act cct 146

Thr Ile Pro Ala Leu Gly Met Ser Arg Arg Gly Lys Ser Ile Thr Pro

35 40 45

tcc atc agc atg agc tct acc acc gtt gta acc gat gat ggt gta cga 194

Ser Ile Ser Met Ser Ser Thr Thr Val Val Thr Asp Asp Gly Val Arg

50 55 60

aga cgc atg ggc gat ttc cat tcc aac ctc tgg gac gat gat gtc ata 242

Arg Arg Met Gly Asp Phe His Ser Asn Leu Trp Asp Asp Asp Val Ile

65 70 75

cag tct tta cca acg gct tat gag gaa aaa tcg tac ctg gag cgt gct 290

Gln Ser Leu Pro Thr Ala Tyr Glu Glu Lys Ser Tyr Leu Glu Arg Ala

80 85 90 95

gag aaa ctg atc ggg gaa gta aag aac atg ttc aat tcg atg tca tta 338

Glu Lys Leu Ile Gly Glu Val Lys Asn Met Phe Asn Ser Met Ser Leu

100 105 110

gaa gat gga gag tta atg agt ccg ctc aat gat ctc att caa cgc ctt 386

Glu Asp Gly Glu Leu Met Ser Pro Leu Asn Asp Leu Ile Gln Arg Leu

115 120 125

tgg att gtc gac agc ctt gaa cgt ttg ggg atc cat aga cat ttc aaa 434

Trp Ile Val Asp Ser Leu Glu Arg Leu Gly Ile His Arg His Phe Lys

130 135 140

gat gag ata aaa tcg gcg ctt gat tat gtt tac agt tat tgg ggc gaa 482

Asp Glu Ile Lys Ser Ala Leu Asp Tyr Val Tyr Ser Tyr Trp Gly Glu

145 150 155

aat ggc atc gga tgc ggg agg gag agt gtt gtt act gat ctg aac tca 530

Asn Gly Ile Gly Cys Gly Arg Glu Ser Val Val Thr Asp Leu Asn Ser

160 165 170 175

act gcg ttg ggg ctt cga acc cta cga cta cac gga tac ccg gtg tct 578

Thr Ala Leu Gly Leu Arg Thr Leu Arg Leu His Gly Tyr Pro Val Ser

180 185 190

tca gat gtt ttc aaa gct ttc aaa ggc caa aat ggg cag ttt tcc tgc 626

Ser Asp Val Phe Lys Ala Phe Lys Gly Gln Asn Gly Gln Phe Ser Cys

195 200 205

tct gaa aat att cag aca gat gaa gag atc aga ggc gtt ctg aat tta 674

Ser Glu Asn Ile Gln Thr Asp Glu Glu Ile Arg Gly Val Leu Asn Leu

210 215 220

ttc cgg gcc tcc ctc att gcc ttt cca ggg gag aaa att atg gat gag 722

Phe Arg Ala Ser Leu Ile Ala Phe Pro Gly Glu Lys Ile Met Asp Glu

225 230 235

gct gaa atc ttc tct acc aaa tat tta aaa gaa gcc ctg caa aag att 770

Ala Glu Ile Phe Ser Thr Lys Tyr Leu Lys Glu Ala Leu Gln Lys Ile

240 245 250 255

ccg gtc tcc agt ctt tcg cga gag atc ggg gac gtt ttg gaa tat ggt 818

Pro Val Ser Ser Leu Ser Arg Glu Ile Gly Asp Val Leu Glu Tyr Gly

260 265 270

tgg cac aca tat ttg ccg cga ttg gaa gca agg aat tac atc caa gtc 866

Trp His Thr Tyr Leu Pro Arg Leu Glu Ala Arg Asn Tyr Ile Gln Val

275 280 285

ttt gga cag gac act gag aac acg aag tca tat gtg aag agc aaa aaa 914

Phe Gly Gln Asp Thr Glu Asn Thr Lys Ser Tyr Val Lys Ser Lys Lys

290 295 300

ctt tta gaa ctc gca aaa ttg gag ttc aac atc ttt caa tcc tta caa 962

Leu Leu Glu Leu Ala Lys Leu Glu Phe Asn Ile Phe Gln Ser Leu Gln

305 310 315

aag agg gag tta gaa agt ctg gtc aga tgg tgg aaa gaa tcg ggt ttt 1010

Lys Arg Glu Leu Glu Ser Leu Val Arg Trp Trp Lys Glu Ser Gly Phe

320 325 330 335

cct gag atg acc ttc tgc cga cat cgt cac gtg gaa tac tac act ttg 1058

Pro Glu Met Thr Phe Cys Arg His Arg His Val Glu Tyr Tyr Thr Leu

340 345 350

gct tcc tgc att gcg ttc gag cct caa cat tct gga ttc aga ctc ggc 1106

Ala Ser Cys Ile Ala Phe Glu Pro Gln His Ser Gly Phe Arg Leu Gly

355 360 365

ttt gcc aag acg tgt cat ctt atc acg gtt ctt gac gat atg tac gac 1154

Phe Ala Lys Thr Cys His Leu Ile Thr Val Leu Asp Asp Met Tyr Asp

370 375 380

acc ttc ggc aca gta gac gag ctg gaa ctc ttc aca gcg aca atg aag 1202

Thr Phe Gly Thr Val Asp Glu Leu Glu Leu Phe Thr Ala Thr Met Lys

385 390 395

aga tgg gat ccg tcc tcg ata gat tgc ctt cca gaa tat atg aaa gga 1250

Arg Trp Asp Pro Ser Ser Ile Asp Cys Leu Pro Glu Tyr Met Lys Gly

400 405 410 415

gtg tac ata gcg gtt tac gac acc gta aat gaa atg gct cga gag gca 1298

Val Tyr Ile Ala Val Tyr Asp Thr Val Asn Glu Met Ala Arg Glu Ala

420 425 430

gag gag gct caa ggc cga gat acg ctc aca tat gct cgg gaa gct tgg 1346

Glu Glu Ala Gln Gly Arg Asp Thr Leu Thr Tyr Ala Arg Glu Ala Trp

435 440 445

gag gct tat att gat tcg tat atg caa gaa gca agg tgg atc gcc act 1394

Glu Ala Tyr Ile Asp Ser Tyr Met Gln Glu Ala Arg Trp Ile Ala Thr

450 455 460

ggt tac ctg ccc tcc ttt gat gag tac tac gag aat ggg aaa gtt agc 1442

Gly Tyr Leu Pro Ser Phe Asp Glu Tyr Tyr Glu Asn Gly Lys Val Ser

465 470 475

tgt ggt cat cgc ata tcc gca ttg caa ccc att ctg aca atg gac atc 1490

Cys Gly His Arg Ile Ser Ala Leu Gln Pro Ile Leu Thr Met Asp Ile

480 485 490 495

ccc ttt cct gat cat atc ctc aag gaa gtt gac ttc cca tca aag ctt 1538

Pro Phe Pro Asp His Ile Leu Lys Glu Val Asp Phe Pro Ser Lys Leu

500 505 510

aac gac ttg gca tgt gcc atc ctt cga tta cga ggt gat acg cgg tgc 1586

Asn Asp Leu Ala Cys Ala Ile Leu Arg Leu Arg Gly Asp Thr Arg Cys

515 520 525

tac aag gcg gac agg gct cgt gga gaa gaa gct tcc tct ata tca tgt 1634

Tyr Lys Ala Asp Arg Ala Arg Gly Glu Glu Ala Ser Ser Ile Ser Cys

530 535 540

tat atg aaa gac aat cct gga gta tca gag gaa gat gct ctc gat cat 1682

Tyr Met Lys Asp Asn Pro Gly Val Ser Glu Glu Asp Ala Leu Asp His

545 550 555

atc aac gcc atg atc agt gac gta atc aaa gga tta aat tgg gaa ctt 1730

Ile Asn Ala Met Ile Ser Asp Val Ile Lys Gly Leu Asn Trp Glu Leu

560 565 570 575

ctc aaa cca gac atc aat gtt ccc atc tcg gcg aag aaa cat gct ttt 1778

Leu Lys Pro Asp Ile Asn Val Pro Ile Ser Ala Lys Lys His Ala Phe

580 585 590

gac atc gcc aga gct ttc cat tac ggc tac aaa tac cga gac ggc tac 1826

Asp Ile Ala Arg Ala Phe His Tyr Gly Tyr Lys Tyr Arg Asp Gly Tyr

595 600 605

agc gtt gcc aac gtt gaa acg aag agt ttg gtc acg aga acc ctc ctt 1874

Ser Val Ala Asn Val Glu Thr Lys Ser Leu Val Thr Arg Thr Leu Leu

610 615 620

gaa tct gtg cct ttg tag caacagctca aatctatgcc ctatgctatg 1922

Glu Ser Val Pro Leu

625

tcgggttaaa atatatgtgg aaggtagccg ttggatgtag aggataagtt tgttataatt 1982

taataaagtt gtaatttaaa aaaaaaaaaa aaaaaa 2018

<210> SEQ ID NO 4

<211> LENGTH: 628

<212> TYPE: PRT

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 4

Met Ala Leu Val Ser Thr Ala Pro Leu Ala Ser Lys Ser Cys Leu His

1 5 10 15

Lys Ser Leu Ile Ser Ser Thr His Glu Leu Lys Ala Leu Ser Arg Thr

20 25 30

Ile Pro Ala Leu Gly Met Ser Arg Arg Gly Lys Ser Ile Thr Pro Ser

35 40 45

Ile Ser Met Ser Ser Thr Thr Val Val Thr Asp Asp Gly Val Arg Arg

50 55 60

Arg Met Gly Asp Phe His Ser Asn Leu Trp Asp Asp Asp Val Ile Gln

65 70 75 80

Ser Leu Pro Thr Ala Tyr Glu Glu Lys Ser Tyr Leu Glu Arg Ala Glu

85 90 95

Lys Leu Ile Gly Glu Val Lys Asn Met Phe Asn Ser Met Ser Leu Glu

100 105 110

Asp Gly Glu Leu Met Ser Pro Leu Asn Asp Leu Ile Gln Arg Leu Trp

115 120 125

Ile Val Asp Ser Leu Glu Arg Leu Gly Ile His Arg His Phe Lys Asp

130 135 140

Glu Ile Lys Ser Ala Leu Asp Tyr Val Tyr Ser Tyr Trp Gly Glu Asn

145 150 155 160

Gly Ile Gly Cys Gly Arg Glu Ser Val Val Thr Asp Leu Asn Ser Thr

165 170 175

Ala Leu Gly Leu Arg Thr Leu Arg Leu His Gly Tyr Pro Val Ser Ser

180 185 190

Asp Val Phe Lys Ala Phe Lys Gly Gln Asn Gly Gln Phe Ser Cys Ser

195 200 205

Glu Asn Ile Gln Thr Asp Glu Glu Ile Arg Gly Val Leu Asn Leu Phe

210 215 220

Arg Ala Ser Leu Ile Ala Phe Pro Gly Glu Lys Ile Met Asp Glu Ala

225 230 235 240

Glu Ile Phe Ser Thr Lys Tyr Leu Lys Glu Ala Leu Gln Lys Ile Pro

245 250 255

Val Ser Ser Leu Ser Arg Glu Ile Gly Asp Val Leu Glu Tyr Gly Trp

260 265 270

His Thr Tyr Leu Pro Arg Leu Glu Ala Arg Asn Tyr Ile Gln Val Phe

275 280 285

Gly Gln Asp Thr Glu Asn Thr Lys Ser Tyr Val Lys Ser Lys Lys Leu

290 295 300

Leu Glu Leu Ala Lys Leu Glu Phe Asn Ile Phe Gln Ser Leu Gln Lys

305 310 315 320

Arg Glu Leu Glu Ser Leu Val Arg Trp Trp Lys Glu Ser Gly Phe Pro

325 330 335

Glu Met Thr Phe Cys Arg His Arg His Val Glu Tyr Tyr Thr Leu Ala

340 345 350

Ser Cys Ile Ala Phe Glu Pro Gln His Ser Gly Phe Arg Leu Gly Phe

355 360 365

Ala Lys Thr Cys His Leu Ile Thr Val Leu Asp Asp Met Tyr Asp Thr

370 375 380

Phe Gly Thr Val Asp Glu Leu Glu Leu Phe Thr Ala Thr Met Lys Arg

385 390 395 400

Trp Asp Pro Ser Ser Ile Asp Cys Leu Pro Glu Tyr Met Lys Gly Val

405 410 415

Tyr Ile Ala Val Tyr Asp Thr Val Asn Glu Met Ala Arg Glu Ala Glu

420 425 430

Glu Ala Gln Gly Arg Asp Thr Leu Thr Tyr Ala Arg Glu Ala Trp Glu

435 440 445

Ala Tyr Ile Asp Ser Tyr Met Gln Glu Ala Arg Trp Ile Ala Thr Gly

450 455 460

Tyr Leu Pro Ser Phe Asp Glu Tyr Tyr Glu Asn Gly Lys Val Ser Cys

465 470 475 480

Gly His Arg Ile Ser Ala Leu Gln Pro Ile Leu Thr Met Asp Ile Pro

485 490 495

Phe Pro Asp His Ile Leu Lys Glu Val Asp Phe Pro Ser Lys Leu Asn

500 505 510

Asp Leu Ala Cys Ala Ile Leu Arg Leu Arg Gly Asp Thr Arg Cys Tyr

515 520 525

Lys Ala Asp Arg Ala Arg Gly Glu Glu Ala Ser Ser Ile Ser Cys Tyr

530 535 540

Met Lys Asp Asn Pro Gly Val Ser Glu Glu Asp Ala Leu Asp His Ile

545 550 555 560

Asn Ala Met Ile Ser Asp Val Ile Lys Gly Leu Asn Trp Glu Leu Leu

565 570 575

Lys Pro Asp Ile Asn Val Pro Ile Ser Ala Lys Lys His Ala Phe Asp

580 585 590

Ile Ala Arg Ala Phe His Tyr Gly Tyr Lys Tyr Arg Asp Gly Tyr Ser

595 600 605

Val Ala Asn Val Glu Thr Lys Ser Leu Val Thr Arg Thr Leu Leu Glu

610 615 620

Ser Val Pro Leu

625

<210> SEQ ID NO 5

<211> LENGTH: 2089

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (73)..(1986)

<223> OTHER INFORMATION:

<400> SEQUENCE: 5

tgccgtttaa tcggtttaaa gaagctacca tagttcggtt taaagaagct accatagttt 60

aggcaggaat cc atg gct ctc ctt tct atc gta tct ttg cag gtt ccc aaa 111

Met Ala Leu Leu Ser Ile Val Ser Leu Gln Val Pro Lys

1 5 10

tcc tgc ggg ctg aaa tcg ttg atc agt tcc agc aat gtg cag aag gct 159

Ser Cys Gly Leu Lys Ser Leu Ile Ser Ser Ser Asn Val Gln Lys Ala

15 20 25

ctc tgt atc tct aca gca gtc cca aca ctc aga atg cgt agg cga cag 207

Leu Cys Ile Ser Thr Ala Val Pro Thr Leu Arg Met Arg Arg Arg Gln

30 35 40 45

aaa gct ctg gtc atc aac atg aaa ttg acc act gta tcc cat cgt gat 255

Lys Ala Leu Val Ile Asn Met Lys Leu Thr Thr Val Ser His Arg Asp

50 55 60

gat aat ggt ggt ggt gta ctg caa aga cgc ata gcc gat cat cat ccc 303

Asp Asn Gly Gly Gly Val Leu Gln Arg Arg Ile Ala Asp His His Pro

65 70 75

aac ctg tgg gaa gat gat ttc ata caa tca ttg tcc tca cct tat ggg 351

Asn Leu Trp Glu Asp Asp Phe Ile Gln Ser Leu Ser Ser Pro Tyr Gly

80 85 90

gga tct tcg tac agt gaa cgt gct gag aca gtc gtt gag gaa gta aaa 399

Gly Ser Ser Tyr Ser Glu Arg Ala Glu Thr Val Val Glu Glu Val Lys

95 100 105

gag atg ttc aat tca ata cca aat aat aga gaa tta ttt ggt tcc caa 447

Glu Met Phe Asn Ser Ile Pro Asn Asn Arg Glu Leu Phe Gly Ser Gln

110 115 120 125

aat gat ctc ctt aca cgc ctt tgg atg gtg gat agc att gaa cgt ctg 495

Asn Asp Leu Leu Thr Arg Leu Trp Met Val Asp Ser Ile Glu Arg Leu

130 135 140

ggg ata gat aga cat ttc caa aat gag ata aga gta gcc ctc gat tat 543

Gly Ile Asp Arg His Phe Gln Asn Glu Ile Arg Val Ala Leu Asp Tyr

145 150 155

gtt tac agt tat tgg aag gaa aag gaa ggc att ggg tgt ggc aga gat 591

Val Tyr Ser Tyr Trp Lys Glu Lys Glu Gly Ile Gly Cys Gly Arg Asp

160 165 170

tct act ttt cct gat ctc aac tcg act gcc ttg gcg ctt cga act ctt 639

Ser Thr Phe Pro Asp Leu Asn Ser Thr Ala Leu Ala Leu Arg Thr Leu

175 180 185

cga ctg cac gga tac aat gtg tct tca gat gtg ctg gaa tac ttc aaa 687

Arg Leu His Gly Tyr Asn Val Ser Ser Asp Val Leu Glu Tyr Phe Lys

190 195 200 205

gat gaa aag ggg cat ttt gcc tgc cct gca atc cta acc gag gga cag 735

Asp Glu Lys Gly His Phe Ala Cys Pro Ala Ile Leu Thr Glu Gly Gln

210 215 220

atc act aga agt gtt cta aat tta tat cgg gct tcc ctg gtc gcc ttt 783

Ile Thr Arg Ser Val Leu Asn Leu Tyr Arg Ala Ser Leu Val Ala Phe

225 230 235

ccc ggg gag aaa gtt atg gaa gag gct gaa atc ttc tcg gca tct tat 831

Pro Gly Glu Lys Val Met Glu Glu Ala Glu Ile Phe Ser Ala Ser Tyr

240 245 250

ttg aaa aaa gtc tta caa aag att ccg gtc tcc aat ctt tca gga gag 879

Leu Lys Lys Val Leu Gln Lys Ile Pro Val Ser Asn Leu Ser Gly Glu

255 260 265

ata gaa tat gtt ttg gaa tat ggt tgg cac acg aat ttg ccg aga ttg 927

Ile Glu Tyr Val Leu Glu Tyr Gly Trp His Thr Asn Leu Pro Arg Leu

270 275 280 285

gaa gca aga aat tat atc gag gtc tac gag cag agc ggc tat gaa agc 975

Glu Ala Arg Asn Tyr Ile Glu Val Tyr Glu Gln Ser Gly Tyr Glu Ser

290 295 300

tta aac gag atg cca tat atg aac atg aag aag ctt tta caa ctt gca 1023

Leu Asn Glu Met Pro Tyr Met Asn Met Lys Lys Leu Leu Gln Leu Ala

305 310 315

aaa ttg gag ttc aat atc ttt cac tct ttg caa cta aga gag tta caa 1071

Lys Leu Glu Phe Asn Ile Phe His Ser Leu Gln Leu Arg Glu Leu Gln

320 325 330

tct atc tcc aga tgg tgg aaa gaa tca ggt tcg tct caa ctg act ttt 1119

Ser Ile Ser Arg Trp Trp Lys Glu Ser Gly Ser Ser Gln Leu Thr Phe

335 340 345

aca cgg cat cgt cac gtg gaa tac tac act atg gca tct tgc att tct 1167

Thr Arg His Arg His Val Glu Tyr Tyr Thr Met Ala Ser Cys Ile Ser

350 355 360 365

atg ttg cca aaa cat tca gct ttc aga atg gag ttt gtc aaa gtg tgt 1215

Met Leu Pro Lys His Ser Ala Phe Arg Met Glu Phe Val Lys Val Cys

370 375 380

cat ctt gta aca gtt ctc gat gat ata tat gac act ttt gga aca atg 1263

His Leu Val Thr Val Leu Asp Asp Ile Tyr Asp Thr Phe Gly Thr Met

385 390 395

aac gaa ctc caa ctt ttt acg gat gca att aag aga tgg gat ttg tca 1311

Asn Glu Leu Gln Leu Phe Thr Asp Ala Ile Lys Arg Trp Asp Leu Ser

400 405 410

acg aca agg tgg ctt cca gaa tat atg aaa gga gtg tac atg gac ttg 1359

Thr Thr Arg Trp Leu Pro Glu Tyr Met Lys Gly Val Tyr Met Asp Leu

415 420 425

tat caa tgc att aat gaa atg gtg gaa gag gct gag aag act caa ggc 1407

Tyr Gln Cys Ile Asn Glu Met Val Glu Glu Ala Glu Lys Thr Gln Gly

430 435 440 445

cga gat atg ctc aac tat att caa aat gct tgg gaa gcc cta ttt gat 1455

Arg Asp Met Leu Asn Tyr Ile Gln Asn Ala Trp Glu Ala Leu Phe Asp

450 455 460

acc ttt atg caa gaa gca aag tgg atc tcc agc agt tat ctc cca acg 1503

Thr Phe Met Gln Glu Ala Lys Trp Ile Ser Ser Ser Tyr Leu Pro Thr

465 470 475

ttt gag gag tac ttg aag aat gca aaa gtt agt tct ggt tct cgc ata 1551

Phe Glu Glu Tyr Leu Lys Asn Ala Lys Val Ser Ser Gly Ser Arg Ile

480 485 490

gcc aca tta caa ccc att ctc act ttg gat gta cca ctt cct gat tac 1599

Ala Thr Leu Gln Pro Ile Leu Thr Leu Asp Val Pro Leu Pro Asp Tyr

495 500 505

ata ctg caa gaa att gat tat cca tcc aga ttc aat gag tta gct tcg 1647

Ile Leu Gln Glu Ile Asp Tyr Pro Ser Arg Phe Asn Glu Leu Ala Ser

510 515 520 525

tcc atc ctt cga cta cga ggt gac acg cgc tgc tac aag gcg gat agg 1695

Ser Ile Leu Arg Leu Arg Gly Asp Thr Arg Cys Tyr Lys Ala Asp Arg

530 535 540

gcc cgt gga gaa gaa gct tca gct ata tcg tgt tat atg aaa gac cat 1743

Ala Arg Gly Glu Glu Ala Ser Ala Ile Ser Cys Tyr Met Lys Asp His

545 550 555

cct gga tca ata gag gaa gat gct ctc aat cat atc aac gcc atg atc 1791

Pro Gly Ser Ile Glu Glu Asp Ala Leu Asn His Ile Asn Ala Met Ile

560 565 570

agt gat gca atc aga gaa tta aat tgg gag ctt ctc aga ccg gat agc 1839

Ser Asp Ala Ile Arg Glu Leu Asn Trp Glu Leu Leu Arg Pro Asp Ser

575 580 585

aaa agt ccc atc tct tcc aag aaa cat gct ttt gac atc acc aga gct 1887

Lys Ser Pro Ile Ser Ser Lys Lys His Ala Phe Asp Ile Thr Arg Ala

590 595 600 605

ttc cat cat gtc tac aaa tat cga gat ggt tac act gtt tcc aac aac 1935

Phe His His Val Tyr Lys Tyr Arg Asp Gly Tyr Thr Val Ser Asn Asn

610 615 620

gaa aca aag aat ttg gtg atg aaa acc gtt ctt gaa cct ctc gct ttg 1983

Glu Thr Lys Asn Leu Val Met Lys Thr Val Leu Glu Pro Leu Ala Leu

625 630 635

taa aaacatatag aatgcattaa aatgtgggaa gtctataatc tagactattc 2036

tctatctttc ataatgtaga tctggatgtg tattgaactc taaaaaaaaa aaa 2089

<210> SEQ ID NO 6

<211> LENGTH: 637

<212> TYPE: PRT

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 6

Met Ala Leu Leu Ser Ile Val Ser Leu Gln Val Pro Lys Ser Cys Gly

1 5 10 15

Leu Lys Ser Leu Ile Ser Ser Ser Asn Val Gln Lys Ala Leu Cys Ile

20 25 30

Ser Thr Ala Val Pro Thr Leu Arg Met Arg Arg Arg Gln Lys Ala Leu

35 40 45

Val Ile Asn Met Lys Leu Thr Thr Val Ser His Arg Asp Asp Asn Gly

50 55 60

Gly Gly Val Leu Gln Arg Arg Ile Ala Asp His His Pro Asn Leu Trp

65 70 75 80

Glu Asp Asp Phe Ile Gln Ser Leu Ser Ser Pro Tyr Gly Gly Ser Ser

85 90 95

Tyr Ser Glu Arg Ala Glu Thr Val Val Glu Glu Val Lys Glu Met Phe

100 105 110

Asn Ser Ile Pro Asn Asn Arg Glu Leu Phe Gly Ser Gln Asn Asp Leu

115 120 125

Leu Thr Arg Leu Trp Met Val Asp Ser Ile Glu Arg Leu Gly Ile Asp

130 135 140

Arg His Phe Gln Asn Glu Ile Arg Val Ala Leu Asp Tyr Val Tyr Ser

145 150 155 160

Tyr Trp Lys Glu Lys Glu Gly Ile Gly Cys Gly Arg Asp Ser Thr Phe

165 170 175

Pro Asp Leu Asn Ser Thr Ala Leu Ala Leu Arg Thr Leu Arg Leu His

180 185 190

Gly Tyr Asn Val Ser Ser Asp Val Leu Glu Tyr Phe Lys Asp Glu Lys

195 200 205

Gly His Phe Ala Cys Pro Ala Ile Leu Thr Glu Gly Gln Ile Thr Arg

210 215 220

Ser Val Leu Asn Leu Tyr Arg Ala Ser Leu Val Ala Phe Pro Gly Glu

225 230 235 240

Lys Val Met Glu Glu Ala Glu Ile Phe Ser Ala Ser Tyr Leu Lys Lys

245 250 255

Val Leu Gln Lys Ile Pro Val Ser Asn Leu Ser Gly Glu Ile Glu Tyr

260 265 270

Val Leu Glu Tyr Gly Trp His Thr Asn Leu Pro Arg Leu Glu Ala Arg

275 280 285

Asn Tyr Ile Glu Val Tyr Glu Gln Ser Gly Tyr Glu Ser Leu Asn Glu

290 295 300

Met Pro Tyr Met Asn Met Lys Lys Leu Leu Gln Leu Ala Lys Leu Glu

305 310 315 320

Phe Asn Ile Phe His Ser Leu Gln Leu Arg Glu Leu Gln Ser Ile Ser

325 330 335

Arg Trp Trp Lys Glu Ser Gly Ser Ser Gln Leu Thr Phe Thr Arg His

340 345 350

Arg His Val Glu Tyr Tyr Thr Met Ala Ser Cys Ile Ser Met Leu Pro

355 360 365

Lys His Ser Ala Phe Arg Met Glu Phe Val Lys Val Cys His Leu Val

370 375 380

Thr Val Leu Asp Asp Ile Tyr Asp Thr Phe Gly Thr Met Asn Glu Leu

385 390 395 400

Gln Leu Phe Thr Asp Ala Ile Lys Arg Trp Asp Leu Ser Thr Thr Arg

405 410 415

Trp Leu Pro Glu Tyr Met Lys Gly Val Tyr Met Asp Leu Tyr Gln Cys

420 425 430

Ile Asn Glu Met Val Glu Glu Ala Glu Lys Thr Gln Gly Arg Asp Met

435 440 445

Leu Asn Tyr Ile Gln Asn Ala Trp Glu Ala Leu Phe Asp Thr Phe Met

450 455 460

Gln Glu Ala Lys Trp Ile Ser Ser Ser Tyr Leu Pro Thr Phe Glu Glu

465 470 475 480

Tyr Leu Lys Asn Ala Lys Val Ser Ser Gly Ser Arg Ile Ala Thr Leu

485 490 495

Gln Pro Ile Leu Thr Leu Asp Val Pro Leu Pro Asp Tyr Ile Leu Gln

500 505 510

Glu Ile Asp Tyr Pro Ser Arg Phe Asn Glu Leu Ala Ser Ser Ile Leu

515 520 525

Arg Leu Arg Gly Asp Thr Arg Cys Tyr Lys Ala Asp Arg Ala Arg Gly

530 535 540

Glu Glu Ala Ser Ala Ile Ser Cys Tyr Met Lys Asp His Pro Gly Ser

545 550 555 560

Ile Glu Glu Asp Ala Leu Asn His Ile Asn Ala Met Ile Ser Asp Ala

565 570 575

Ile Arg Glu Leu Asn Trp Glu Leu Leu Arg Pro Asp Ser Lys Ser Pro

580 585 590

Ile Ser Ser Lys Lys His Ala Phe Asp Ile Thr Arg Ala Phe His His

595 600 605

Val Tyr Lys Tyr Arg Asp Gly Tyr Thr Val Ser Asn Asn Glu Thr Lys

610 615 620

Asn Leu Val Met Lys Thr Val Leu Glu Pro Leu Ala Leu

625 630 635

<210> SEQ ID NO 7

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Degenerate oligonucleotide PCR primer A

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(25)

<223> OTHER INFORMATION: Wherein N=inosine

<400> SEQUENCE: 7

arraygarra nggnrartay aarga 25

<210> SEQ ID NO 8

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Degenerate oligonucleotide PCR primer B

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(20)

<223> OTHER INFORMATION: Wherein N=inosine residue

<400> SEQUENCE: 8

atgytncary tntaygargc 20

<210> SEQ ID NO 9

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Degenerate oligonucleotide PCR primer C

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(24)

<223> OTHER INFORMATION: Wherein N=inosine residue

<400> SEQUENCE: 9

ctnkynrang gnctratrta ckty 24

<210> SEQ ID NO 10

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Degenerate oligonucleotide PCR primer D

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(23)

<223> OTHER INFORMATION: Wherein N=isonine

<400> SEQUENCE: 10

gaygaynnnt wygaygcnya ygg 23

<210> SEQ ID NO 11

<211> LENGTH: 108

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 11

gatgatgggt ttgatgcgca cggaacccta gatgaattga agctattcac tgaggctgtg 60

agaagatggg acctctcctt tacagacaac ttccccgatt acatgaaa 108

<210> SEQ ID NO 12

<211> LENGTH: 104

TYPE: DNA

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 12

gacgacgggt atgatgcgca tggaacgatt gacgagcttg aactcttcac atctgcaatt 60

aagagatgga attcatcaga gatagacagc ttccccgact atat 104

<210> SEQ ID NO 13

<211> LENGTH: 105

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(105)

<223> OTHER INFORMATION: Wherein N=A, C, G or T

<400> SEQUENCE: 13

gatgatgggt atgatgcgta cggaacgttg gaagaaatca aaatcatgac agagggagtg 60

agacgatggg atctttcgtt gaccgcttnc cccgactata tgaaa 105

<210> SEQ ID NO 14

<211> LENGTH: 117

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(117)

<223> OTHER INFORMATION: Wherein N=A, C, G or T

<400> SEQUENCE: 14

gacgatgggt atgatgcgca tggaaccttg gaccaactca aaatctttac agagggagtg 60

agacgatggg atgtttcgtt ggtagaccac ttnccccgac tacatgcaat ctagacc 117

<210> SEQ ID NO 15

<211> LENGTH: 2424

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (2)..(2350)

<223> OTHER INFORMATION:

<400> SEQUENCE: 15

g ggt tat gat ctt gtg cat tct ctt aaa tca cct tat att gat tct agt 49

Gly Tyr Asp Leu Val His Ser Leu Lys Ser Pro Tyr Ile Asp Ser Ser

1 5 10 15

tac aga gaa cgc gcg gag gtc ctt gtt agc gag att aaa gtg atg ctt 97

Tyr Arg Glu Arg Ala Glu Val Leu Val Ser Glu Ile Lys Val Met Leu

20 25 30

aat cca gct att aca gga gat gga gaa tca atg att act cca tct gct 145

Asn Pro Ala Ile Thr Gly Asp Gly Glu Ser Met Ile Thr Pro Ser Ala

35 40 45

tat gac aca gca tgg gta gcg agg gtg ccc gcc att gat ggc tct gct 193

Tyr Asp Thr Ala Trp Val Ala Arg Val Pro Ala Ile Asp Gly Ser Ala

50 55 60

cgc ccg caa ttt ccc caa aca gtt gac tgg att ttg aaa aac cag tta 241

Arg Pro Gln Phe Pro Gln Thr Val Asp Trp Ile Leu Lys Asn Gln Leu

65 70 75 80

aaa gat ggt tca tgg gga att cag tcc cac ttt ctg ctg tcc gac cgt 289

Lys Asp Gly Ser Trp Gly Ile Gln Ser His Phe Leu Leu Ser Asp Arg

85 90 95

ctt ctt gcc act ctt tct tgt gtt ctt gtg ctc ctt aaa tgg aac gtt 337

Leu Leu Ala Thr Leu Ser Cys Val Leu Val Leu Leu Lys Trp Asn Val

100 105 110

ggg gat ctg caa gta gag cag gga att gaa ttc ata aag agc aat ctg 385

Gly Asp Leu Gln Val Glu Gln Gly Ile Glu Phe Ile Lys Ser Asn Leu

115 120 125

gaa cta gta aag gat gaa acc gat caa gat agc ttg gta aca gac ttt 433

Glu Leu Val Lys Asp Glu Thr Asp Gln Asp Ser Leu Val Thr Asp Phe

130 135 140

gag atc ata ttt cct tct ctg tta aga gaa gct caa tct ctg cgc ctc 481

Glu Ile Ile Phe Pro Ser Leu Leu Arg Glu Ala Gln Ser Leu Arg Leu

145 150 155 160

gga ctt ccc tac gac ctg cct tat ata cat ctg ttg cag act aaa cgg 529

Gly Leu Pro Tyr Asp Leu Pro Tyr Ile His Leu Leu Gln Thr Lys Arg

165 170 175

cag gaa aga tta gca aaa ctt tca agg gag gaa att tat gcg gtt ccg 577

Gln Glu Arg Leu Ala Lys Leu Ser Arg Glu Glu Ile Tyr Ala Val Pro

180 185 190

tcg cca ttg ttg tat tct tta gag gga ata caa gat ata gtt gaa tgg 625

Ser Pro Leu Leu Tyr Ser Leu Glu Gly Ile Gln Asp Ile Val Glu Trp

195 200 205

gaa cga ata atg gaa gtt caa agt cag gat ggg tct ttc tta agc tca 673

Glu Arg Ile Met Glu Val Gln Ser Gln Asp Gly Ser Phe Leu Ser Ser

210 215 220

cct gct tct act gcc tgc gtt ttc atg cac aca gga gac gcg aaa tgc 721

Pro Ala Ser Thr Ala Cys Val Phe Met His Thr Gly Asp Ala Lys Cys

225 230 235 240

ctt gaa ttc ttg aac agt gtg atg atc aag ttt gga aat ttt gtt ccc 769

Leu Glu Phe Leu Asn Ser Val Met Ile Lys Phe Gly Asn Phe Val Pro

245 250 255

tgc ctg tat cct gtg gat ctg ctg gaa cgc ctg ttg atc gta gat aat 817

Cys Leu Tyr Pro Val Asp Leu Leu Glu Arg Leu Leu Ile Val Asp Asn

260 265 270

att gta cgc ctt gga atc tat aga cac ttt gaa aag gaa atc aag gaa 865

Ile Val Arg Leu Gly Ile Tyr Arg His Phe Glu Lys Glu Ile Lys Glu

275 280 285

gct ctt gat tat gtt tac agg cat tgg aac gaa aga gga att ggg tgg 913

Ala Leu Asp Tyr Val Tyr Arg His Trp Asn Glu Arg Gly Ile Gly Trp

290 295 300

ggc aga cta aat ccc ata gca gat ctt gag acc act gct ttg gga ttt 961

Gly Arg Leu Asn Pro Ile Ala Asp Leu Glu Thr Thr Ala Leu Gly Phe

305 310 315 320

cga ttg ctt cgg ctg cat agg tac aat gta tct cca gcc att ttt gac 1009

Arg Leu Leu Arg Leu His Arg Tyr Asn Val Ser Pro Ala Ile Phe Asp

325 330 335

aac ttc aaa gat gcc aat ggg aaa ttc att tgc tcg acc ggt caa ttc 1057

Asn Phe Lys Asp Ala Asn Gly Lys Phe Ile Cys Ser Thr Gly Gln Phe

340 345 350

aac aaa gat gta gca agc atg ctg aat ctt tat aga gct tcc cag ctc 1105

Asn Lys Asp Val Ala Ser Met Leu Asn Leu Tyr Arg Ala Ser Gln Leu

355 360 365

gca ttt ccc gga gaa aac att ctt gat gaa gct aaa agc ttc gct act 1153

Ala Phe Pro Gly Glu Asn Ile Leu Asp Glu Ala Lys Ser Phe Ala Thr

370 375 380

aaa tat ttg aga gaa gct ctt gag aaa agt gag act tcc agt gca tgg 1201

Lys Tyr Leu Arg Glu Ala Leu Glu Lys Ser Glu Thr Ser Ser Ala Trp

385 390 395 400

aac aac aaa caa aac ctg agc caa gag atc aaa tac gcg ctg aag act 1249

Asn Asn Lys Gln Asn Leu Ser Gln Glu Ile Lys Tyr Ala Leu Lys Thr

405 410 415

tct tgg cat gcc agt gtt ccg aga gtg gaa gca aag aga tac tgt caa 1297

Ser Trp His Ala Ser Val Pro Arg Val Glu Ala Lys Arg Tyr Cys Gln

420 425 430

gtg tat cgc cca gat tat gca cgc ata gca aaa tgc gtt tac aag cta 1345

Val Tyr Arg Pro Asp Tyr Ala Arg Ile Ala Lys Cys Val Tyr Lys Leu

435 440 445

ccc tac gtg aac aat gaa aag ttt tta gag ctg gga aaa tta gat ttc 1393

Pro Tyr Val Asn Asn Glu Lys Phe Leu Glu Leu Gly Lys Leu Asp Phe

450 455 460

aac att atc cag tcc atc cac caa gaa gaa atg aag aat gtt acc agc 1441

Asn Ile Ile Gln Ser Ile His Gln Glu Glu Met Lys Asn Val Thr Ser

465 470 475 480

tgg ttt aga gat tcg ggg ttg cca cta ttc acc ttc gct cgg gag agg 1489

Trp Phe Arg Asp Ser Gly Leu Pro Leu Phe Thr Phe Ala Arg Glu Arg

485 490 495

ccg ctg gaa ttc tac ttc tta gta gcg gcg ggg acc tat gaa ccc cag 1537

Pro Leu Glu Phe Tyr Phe Leu Val Ala Ala Gly Thr Tyr Glu Pro Gln

500 505 510

tat gcc aaa tgc agg ttc ctc ttt aca aaa gtg gca tgc ttg cag act 1585

Tyr Ala Lys Cys Arg Phe Leu Phe Thr Lys Val Ala Cys Leu Gln Thr

515 520 525

gtt ctg gac gat atg tat gac act tat gga acc cta gat gaa ttg aag 1633

Val Leu Asp Asp Met Tyr Asp Thr Tyr Gly Thr Leu Asp Glu Leu Lys

530 535 540

cta ttc act gag gct gtg aga aga tgg gac ctc tcc ttt aca gaa aac 1681

Leu Phe Thr Glu Ala Val Arg Arg Trp Asp Leu Ser Phe Thr Glu Asn

545 550 555 560

ctt cca gac tat atg aaa cta tgt tac caa atc tat tat gac ata gtt 1729

Leu Pro Asp Tyr Met Lys Leu Cys Tyr Gln Ile Tyr Tyr Asp Ile Val

565 570 575

cac gag gtg gct tgg gag gca gag aag gaa cag ggg cgt gaa ttg gtc 1777

His Glu Val Ala Trp Glu Ala Glu Lys Glu Gln Gly Arg Glu Leu Val

580 585 590

agc ttt ttc aga aag gga tgg gag gat tat ctt ctg ggt tat tat gaa 1825

Ser Phe Phe Arg Lys Gly Trp Glu Asp Tyr Leu Leu Gly Tyr Tyr Glu

595 600 605

gaa gct gaa tgg tta gct gct gag tat gtg cct acc ttg gac gag tac 1873

Glu Ala Glu Trp Leu Ala Ala Glu Tyr Val Pro Thr Leu Asp Glu Tyr

610 615 620

ata aag aat gga atc aca tct atc ggc caa cgt ata ctt ctg ttg agt 1921

Ile Lys Asn Gly Ile Thr Ser Ile Gly Gln Arg Ile Leu Leu Leu Ser

625 630 635 640

gga gtg ttg ata atg gat ggg caa ctc ctt tcg caa gag gca tta gag 1969

Gly Val Leu Ile Met Asp Gly Gln Leu Leu Ser Gln Glu Ala Leu Glu

645 650 655

aaa gta gat tat cca gga aga cgt gtt ctc aca gag ctg aat agc ctc 2017

Lys Val Asp Tyr Pro Gly Arg Arg Val Leu Thr Glu Leu Asn Ser Leu

660 665 670

att tcc cgc ctg gcg gat gac acg aag aca tat aaa gct gag aag gct 2065

Ile Ser Arg Leu Ala Asp Asp Thr Lys Thr Tyr Lys Ala Glu Lys Ala

675 680 685

cgt gga gaa ttg gcg tcc agc att gaa tgt tac atg aaa gac cat cct 2113

Arg Gly Glu Leu Ala Ser Ser Ile Glu Cys Tyr Met Lys Asp His Pro

690 695 700

gaa tgt aca gag gaa gag gct ctc gat cac atc tat agc att ctg gag 2161

Glu Cys Thr Glu Glu Glu Ala Leu Asp His Ile Tyr Ser Ile Leu Glu

705 710 715 720

ccg gcg gtg aag gaa ctg aca aga gag ttt ctg aag ccc gac gac gtc 2209

Pro Ala Val Lys Glu Leu Thr Arg Glu Phe Leu Lys Pro Asp Asp Val

725 730 735

cca ttc gcc tgc aag aag atg ctt ttc gag gag aca aga gtg acg atg 2257

Pro Phe Ala Cys Lys Lys Met Leu Phe Glu Glu Thr Arg Val Thr Met

740 745 750

gtg ata ttc aag gat gga gat gga ttc ggt gtt tcc aaa tta gaa gtc 2305

Val Ile Phe Lys Asp Gly Asp Gly Phe Gly Val Ser Lys Leu Glu Val

755 760 765

aaa gat cat atc aaa gag tgt ctc att gaa ccg ctg cca ctg taa 2350

Lys Asp His Ile Lys Glu Cys Leu Ile Glu Pro Leu Pro Leu

770 775 780

tcaaaatagt tgcaataata attgaaataa tgtcaactat gtttcacaaa aaaaaaaaaa 2410

aaaaaaaaaa aaaa 2424

<210> SEQ ID NO 16

<211> LENGTH: 782

<212> TYPE: PRT

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 16

Gly Tyr Asp Leu Val His Ser Leu Lys Ser Pro Tyr Ile Asp Ser Ser

1 5 10 15

Tyr Arg Glu Arg Ala Glu Val Leu Val Ser Glu Ile Lys Val Met Leu

20 25 30

Asn Pro Ala Ile Thr Gly Asp Gly Glu Ser Met Ile Thr Pro Ser Ala

35 40 45

Tyr Asp Thr Ala Trp Val Ala Arg Val Pro Ala Ile Asp Gly Ser Ala

50 55 60

Arg Pro Gln Phe Pro Gln Thr Val Asp Trp Ile Leu Lys Asn Gln Leu

65 70 75 80

Lys Asp Gly Ser Trp Gly Ile Gln Ser His Phe Leu Leu Ser Asp Arg

85 90 95

Leu Leu Ala Thr Leu Ser Cys Val Leu Val Leu Leu Lys Trp Asn Val

100 105 110

Gly Asp Leu Gln Val Glu Gln Gly Ile Glu Phe Ile Lys Ser Asn Leu

115 120 125

Glu Leu Val Lys Asp Glu Thr Asp Gln Asp Ser Leu Val Thr Asp Phe

130 135 140

Glu Ile Ile Phe Pro Ser Leu Leu Arg Glu Ala Gln Ser Leu Arg Leu

145 150 155 160

Gly Leu Pro Tyr Asp Leu Pro Tyr Ile His Leu Leu Gln Thr Lys Arg

165 170 175

Gln Glu Arg Leu Ala Lys Leu Ser Arg Glu Glu Ile Tyr Ala Val Pro

180 185 190

Ser Pro Leu Leu Tyr Ser Leu Glu Gly Ile Gln Asp Ile Val Glu Trp

195 200 205

Glu Arg Ile Met Glu Val Gln Ser Gln Asp Gly Ser Phe Leu Ser Ser

210 215 220

Pro Ala Ser Thr Ala Cys Val Phe Met His Thr Gly Asp Ala Lys Cys

225 230 235 240

Leu Glu Phe Leu Asn Ser Val Met Ile Lys Phe Gly Asn Phe Val Pro

245 250 255

Cys Leu Tyr Pro Val Asp Leu Leu Glu Arg Leu Leu Ile Val Asp Asn

260 265 270

Ile Val Arg Leu Gly Ile Tyr Arg His Phe Glu Lys Glu Ile Lys Glu

275 280 285

Ala Leu Asp Tyr Val Tyr Arg His Trp Asn Glu Arg Gly Ile Gly Trp

290 295 300

Gly Arg Leu Asn Pro Ile Ala Asp Leu Glu Thr Thr Ala Leu Gly Phe

305 310 315 320

Arg Leu Leu Arg Leu His Arg Tyr Asn Val Ser Pro Ala Ile Phe Asp

325 330 335

Asn Phe Lys Asp Ala Asn Gly Lys Phe Ile Cys Ser Thr Gly Gln Phe

340 345 350

Asn Lys Asp Val Ala Ser Met Leu Asn Leu Tyr Arg Ala Ser Gln Leu

355 360 365

Ala Phe Pro Gly Glu Asn Ile Leu Asp Glu Ala Lys Ser Phe Ala Thr

370 375 380

Lys Tyr Leu Arg Glu Ala Leu Glu Lys Ser Glu Thr Ser Ser Ala Trp

385 390 395 400

Asn Asn Lys Gln Asn Leu Ser Gln Glu Ile Lys Tyr Ala Leu Lys Thr

405 410 415

Ser Trp His Ala Ser Val Pro Arg Val Glu Ala Lys Arg Tyr Cys Gln

420 425 430

Val Tyr Arg Pro Asp Tyr Ala Arg Ile Ala Lys Cys Val Tyr Lys Leu

435 440 445

Pro Tyr Val Asn Asn Glu Lys Phe Leu Glu Leu Gly Lys Leu Asp Phe

450 455 460

Asn Ile Ile Gln Ser Ile His Gln Glu Glu Met Lys Asn Val Thr Ser

465 470 475 480

Trp Phe Arg Asp Ser Gly Leu Pro Leu Phe Thr Phe Ala Arg Glu Arg

485 490 495

Pro Leu Glu Phe Tyr Phe Leu Val Ala Ala Gly Thr Tyr Glu Pro Gln

500 505 510

Tyr Ala Lys Cys Arg Phe Leu Phe Thr Lys Val Ala Cys Leu Gln Thr

515 520 525

Val Leu Asp Asp Met Tyr Asp Thr Tyr Gly Thr Leu Asp Glu Leu Lys

530 535 540

Leu Phe Thr Glu Ala Val Arg Arg Trp Asp Leu Ser Phe Thr Glu Asn

545 550 555 560

Leu Pro Asp Tyr Met Lys Leu Cys Tyr Gln Ile Tyr Tyr Asp Ile Val

565 570 575

His Glu Val Ala Trp Glu Ala Glu Lys Glu Gln Gly Arg Glu Leu Val

580 585 590

Ser Phe Phe Arg Lys Gly Trp Glu Asp Tyr Leu Leu Gly Tyr Tyr Glu

595 600 605

Glu Ala Glu Trp Leu Ala Ala Glu Tyr Val Pro Thr Leu Asp Glu Tyr

610 615 620

Ile Lys Asn Gly Ile Thr Ser Ile Gly Gln Arg Ile Leu Leu Leu Ser

625 630 635 640

Gly Val Leu Ile Met Asp Gly Gln Leu Leu Ser Gln Glu Ala Leu Glu

645 650 655

Lys Val Asp Tyr Pro Gly Arg Arg Val Leu Thr Glu Leu Asn Ser Leu

660 665 670

Ile Ser Arg Leu Ala Asp Asp Thr Lys Thr Tyr Lys Ala Glu Lys Ala

675 680 685

Arg Gly Glu Leu Ala Ser Ser Ile Glu Cys Tyr Met Lys Asp His Pro

690 695 700

Glu Cys Thr Glu Glu Glu Ala Leu Asp His Ile Tyr Ser Ile Leu Glu

705 710 715 720

Pro Ala Val Lys Glu Leu Thr Arg Glu Phe Leu Lys Pro Asp Asp Val

725 730 735

Pro Phe Ala Cys Lys Lys Met Leu Phe Glu Glu Thr Arg Val Thr Met

740 745 750

Val Ile Phe Lys Asp Gly Asp Gly Phe Gly Val Ser Lys Leu Glu Val

755 760 765

Lys Asp His Ile Lys Glu Cys Leu Ile Glu Pro Leu Pro Leu

770 775 780

<210> SEQ ID NO 17

<211> LENGTH: 1967

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (3)..(1736)

<223> OTHER INFORMATION:

<400> SEQUENCE: 17

tt tct gaa tct tcc atc cct cga cgc aca ggg aat cat cac gga aat 47

Ser Glu Ser Ser Ile Pro Arg Arg Thr Gly Asn His His Gly Asn

1 5 10 15

gtg tgg gac gat gac ctc ata cac tct ctc aac tcg ccc tat ggg gca 95

Val Trp Asp Asp Asp Leu Ile His Ser Leu Asn Ser Pro Tyr Gly Ala

20 25 30

cct gca tat tat gag ctc ctt caa aag ctt att gag gag atc aag cat 143

Pro Ala Tyr Tyr Glu Leu Leu Gln Lys Leu Ile Glu Glu Ile Lys His

35 40 45

tta ctt ttg act gaa atg gaa atg gat gat ggc gat cat gat tta atc 191

Leu Leu Leu Thr Glu Met Glu Met Asp Asp Gly Asp His Asp Leu Ile

50 55 60

aaa cgt ctt cag atc gtt gac act ttg gaa tgc ctg gga atc gat aga 239

Lys Arg Leu Gln Ile Val Asp Thr Leu Glu Cys Leu Gly Ile Asp Arg

65 70 75

cat ttt gaa cac gaa ata caa aca gct gct tta gat tac gtt tac aga 287

His Phe Glu His Glu Ile Gln Thr Ala Ala Leu Asp Tyr Val Tyr Arg

80 85 90 95

tgg tgg aac gaa aaa ggt atc ggg gag gga tca aga gat tcc ttc agc 335

Trp Trp Asn Glu Lys Gly Ile Gly Glu Gly Ser Arg Asp Ser Phe Ser

100 105 110

aaa gat ctc aac gct aca gct tta gga ttt cgc gct ctc cga ctg cat 383

Lys Asp Leu Asn Ala Thr Ala Leu Gly Phe Arg Ala Leu Arg Leu His

115 120 125

cga tat aac gta tcg tca ggt gtg ttg aag aat ttc aag gat gaa aac 431

Arg Tyr Asn Val Ser Ser Gly Val Leu Lys Asn Phe Lys Asp Glu Asn

130 135 140

ggg aag ttc ttc tgc aac ttt act ggt gaa gaa gga aga gga gat aaa 479

Gly Lys Phe Phe Cys Asn Phe Thr Gly Glu Glu Gly Arg Gly Asp Lys

145 150 155

caa gtg aga agc atg ttg tcg tta ctt cga gct tca gag att tcg ttt 527

Gln Val Arg Ser Met Leu Ser Leu Leu Arg Ala Ser Glu Ile Ser Phe

160 165 170 175

ccc gga gaa aaa gtg atg gaa gag gcc aag gca ttc aca aga gaa tat 575

Pro Gly Glu Lys Val Met Glu Glu Ala Lys Ala Phe Thr Arg Glu Tyr

180 185 190

cta aac caa gtt tta gct gga cac ggg gat gtg act gac gtg gat caa 623

Leu Asn Gln Val Leu Ala Gly His Gly Asp Val Thr Asp Val Asp Gln

195 200 205

agc ctt ttg gag aga ggt gaa gta cgc att gga gtt tcc atg gct tgc 671

Ser Leu Leu Glu Arg Gly Glu Val Arg Ile Gly Val Ser Met Ala Cys

210 215 220

agt gtg ccg aga tgg gag gca agg agc ttt ctc gaa ata tat gga cac 719

Ser Val Pro Arg Trp Glu Ala Arg Ser Phe Leu Glu Ile Tyr Gly His

225 230 235

aac cat tcg tgg ctc aag tcg aat atc aac caa aaa atg ttg aag tta 767

Asn His Ser Trp Leu Lys Ser Asn Ile Asn Gln Lys Met Leu Lys Leu

240 245 250 255

gcc aaa ttg gac ttc aat att ctg caa tgc aaa cat cac aag gag ata 815

Ala Lys Leu Asp Phe Asn Ile Leu Gln Cys Lys His His Lys Glu Ile

260 265 270

cag ttt att aca agg tgg tgg aga gac tcg ggt ata tcg cag ctg aat 863

Gln Phe Ile Thr Arg Trp Trp Arg Asp Ser Gly Ile Ser Gln Leu Asn

275 280 285

ttc tat cga aag cga cac gtg gaa tat tat tct tgg gtt gtt atg tgc 911

Phe Tyr Arg Lys Arg His Val Glu Tyr Tyr Ser Trp Val Val Met Cys

290 295 300

att ttt gag cca gag ttc tct gaa agt aga att gcc ttc gcc aaa act 959

Ile Phe Glu Pro Glu Phe Ser Glu Ser Arg Ile Ala Phe Ala Lys Thr

305 310 315

gct atc cta tgt act gtt cta gat gac ctc tat gat acg cac gca acg 1007

Ala Ile Leu Cys Thr Val Leu Asp Asp Leu Tyr Asp Thr His Ala Thr

320 325 330 335

ttg cat gaa atc aaa atc atg aca gag gga gtg aga cga tgg gat ctt 1055

Leu His Glu Ile Lys Ile Met Thr Glu Gly Val Arg Arg Trp Asp Leu

340 345 350

tcg ttg aca gat gac ctc cca gac tac att aaa att gca ttc cag ttc 1103

Ser Leu Thr Asp Asp Leu Pro Asp Tyr Ile Lys Ile Ala Phe Gln Phe

355 360 365

ttc ttc aat aca gtg aat gaa ttg ata gtt gaa atc gtg aaa cgg caa 1151

Phe Phe Asn Thr Val Asn Glu Leu Ile Val Glu Ile Val Lys Arg Gln

370 375 380

ggg cgg gat atg aca acc ata gtt aaa gat tgc tgg aag cga tac att 1199

Gly Arg Asp Met Thr Thr Ile Val Lys Asp Cys Trp Lys Arg Tyr Ile

385 390 395

gag tct tat ctg caa gaa gcg gaa tgg ata gca act gga cat att ccc 1247

Glu Ser Tyr Leu Gln Glu Ala Glu Trp Ile Ala Thr Gly His Ile Pro

400 405 410 415

act ttt aac gaa tac ata aag aac ggc atg gct agc tca ggg atg tgt 1295

Thr Phe Asn Glu Tyr Ile Lys Asn Gly Met Ala Ser Ser Gly Met Cys

420 425 430

att gta aat ttg aat cca ctt ctc ttg ttg ggt aaa ctt ctc ccc gac 1343

Ile Val Asn Leu Asn Pro Leu Leu Leu Leu Gly Lys Leu Leu Pro Asp

435 440 445

aac att ctg gag caa ata cat tct cca tcc aag atc ctg gac ctc tta 1391

Asn Ile Leu Glu Gln Ile His Ser Pro Ser Lys Ile Leu Asp Leu Leu

450 455 460

gaa ttg acg ggc aga atc gcc gat gac tta aaa gat ttc gag gac gag 1439

Glu Leu Thr Gly Arg Ile Ala Asp Asp Leu Lys Asp Phe Glu Asp Glu

465 470 475

aag gaa cgc ggg gag atg gct tca tct tta cag tgt tat atg aaa gaa 1487

Lys Glu Arg Gly Glu Met Ala Ser Ser Leu Gln Cys Tyr Met Lys Glu

480 485 490 495

aat cct gaa tct aca gtg gaa aat gct tta aat cac ata aaa ggc atc 1535

Asn Pro Glu Ser Thr Val Glu Asn Ala Leu Asn His Ile Lys Gly Ile

500 505 510

ctt aat cgt tcc ctt gag gaa ttt aat tgg gag ttt atg aag cag gat 1583

Leu Asn Arg Ser Leu Glu Glu Phe Asn Trp Glu Phe Met Lys Gln Asp

515 520 525

agt gtc cca atg tgt tgc aag aaa ttc act ttc aat ata ggt cga gga 1631

Ser Val Pro Met Cys Cys Lys Lys Phe Thr Phe Asn Ile Gly Arg Gly

530 535 540

ctt caa ttc atc tac aaa tac aga gac ggc tta tac att tct gac aag 1679

Leu Gln Phe Ile Tyr Lys Tyr Arg Asp Gly Leu Tyr Ile Ser Asp Lys

545 550 555

gaa gta aag gac cag ata ttc aaa att cta gtc cac caa gtt cca atg 1727

Glu Val Lys Asp Gln Ile Phe Lys Ile Leu Val His Gln Val Pro Met

560 565 570 575

gag gaa tag tgatggtctt ggttgtagtt gtctattatg gtatattgca 1776

Glu Glu

ttgacattta tgcttaaagg tgtttcttaa acgtttaggg cggaccgtta aataagttgg 1836

caataattaa tatttagaga ctttgtggaa gtgtttaggg cataaaattg cctatggcct 1896

atggcaagct acaaattgaa attgttgtgt ttataatatt tttattttat ttaaaaaaaa 1956

aaaaaaaaaa a 1967

<210> SEQ ID NO 18

<211> LENGTH: 577

<212> TYPE: PRT

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 18

Ser Glu Ser Ser Ile Pro Arg Arg Thr Gly Asn His His Gly Asn Val

1 5 10 15

Trp Asp Asp Asp Leu Ile His Ser Leu Asn Ser Pro Tyr Gly Ala Pro

20 25 30

Ala Tyr Tyr Glu Leu Leu Gln Lys Leu Ile Glu Glu Ile Lys His Leu

35 40 45

Leu Leu Thr Glu Met Glu Met Asp Asp Gly Asp His Asp Leu Ile Lys

50 55 60

Arg Leu Gln Ile Val Asp Thr Leu Glu Cys Leu Gly Ile Asp Arg His

65 70 75 80

Phe Glu His Glu Ile Gln Thr Ala Ala Leu Asp Tyr Val Tyr Arg Trp

85 90 95

Trp Asn Glu Lys Gly Ile Gly Glu Gly Ser Arg Asp Ser Phe Ser Lys

100 105 110

Asp Leu Asn Ala Thr Ala Leu Gly Phe Arg Ala Leu Arg Leu His Arg

115 120 125

Tyr Asn Val Ser Ser Gly Val Leu Lys Asn Phe Lys Asp Glu Asn Gly

130 135 140

Lys Phe Phe Cys Asn Phe Thr Gly Glu Glu Gly Arg Gly Asp Lys Gln

145 150 155 160

Val Arg Ser Met Leu Ser Leu Leu Arg Ala Ser Glu Ile Ser Phe Pro

165 170 175

Gly Glu Lys Val Met Glu Glu Ala Lys Ala Phe Thr Arg Glu Tyr Leu

180 185 190

Asn Gln Val Leu Ala Gly His Gly Asp Val Thr Asp Val Asp Gln Ser

195 200 205

Leu Leu Glu Arg Gly Glu Val Arg Ile Gly Val Ser Met Ala Cys Ser

210 215 220

Val Pro Arg Trp Glu Ala Arg Ser Phe Leu Glu Ile Tyr Gly His Asn

225 230 235 240

His Ser Trp Leu Lys Ser Asn Ile Asn Gln Lys Met Leu Lys Leu Ala

245 250 255

Lys Leu Asp Phe Asn Ile Leu Gln Cys Lys His His Lys Glu Ile Gln

260 265 270

Phe Ile Thr Arg Trp Trp Arg Asp Ser Gly Ile Ser Gln Leu Asn Phe

275 280 285

Tyr Arg Lys Arg His Val Glu Tyr Tyr Ser Trp Val Val Met Cys Ile

290 295 300

Phe Glu Pro Glu Phe Ser Glu Ser Arg Ile Ala Phe Ala Lys Thr Ala

305 310 315 320

Ile Leu Cys Thr Val Leu Asp Asp Leu Tyr Asp Thr His Ala Thr Leu

325 330 335

His Glu Ile Lys Ile Met Thr Glu Gly Val Arg Arg Trp Asp Leu Ser

340 345 350

Leu Thr Asp Asp Leu Pro Asp Tyr Ile Lys Ile Ala Phe Gln Phe Phe

355 360 365

Phe Asn Thr Val Asn Glu Leu Ile Val Glu Ile Val Lys Arg Gln Gly

370 375 380

Arg Asp Met Thr Thr Ile Val Lys Asp Cys Trp Lys Arg Tyr Ile Glu

385 390 395 400

Ser Tyr Leu Gln Glu Ala Glu Trp Ile Ala Thr Gly His Ile Pro Thr

405 410 415

Phe Asn Glu Tyr Ile Lys Asn Gly Met Ala Ser Ser Gly Met Cys Ile

420 425 430

Val Asn Leu Asn Pro Leu Leu Leu Leu Gly Lys Leu Leu Pro Asp Asn

435 440 445

Ile Leu Glu Gln Ile His Ser Pro Ser Lys Ile Leu Asp Leu Leu Glu

450 455 460

Leu Thr Gly Arg Ile Ala Asp Asp Leu Lys Asp Phe Glu Asp Glu Lys

465 470 475 480

Glu Arg Gly Glu Met Ala Ser Ser Leu Gln Cys Tyr Met Lys Glu Asn

485 490 495

Pro Glu Ser Thr Val Glu Asn Ala Leu Asn His Ile Lys Gly Ile Leu

500 505 510

Asn Arg Ser Leu Glu Glu Phe Asn Trp Glu Phe Met Lys Gln Asp Ser

515 520 525

Val Pro Met Cys Cys Lys Lys Phe Thr Phe Asn Ile Gly Arg Gly Leu

530 535 540

Gln Phe Ile Tyr Lys Tyr Arg Asp Gly Leu Tyr Ile Ser Asp Lys Glu

545 550 555 560

Val Lys Asp Gln Ile Phe Lys Ile Leu Val His Gln Val Pro Met Glu

565 570 575

Glu

<210> SEQ ID NO 19

<211> LENGTH: 1416

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (3)..(1199)

<223> OTHER INFORMATION:

<400> SEQUENCE: 19

aa aaa gtg atg gaa gag gcg aag gca ttc aca aca aat tat cta aag 47

Lys Val Met Glu Glu Ala Lys Ala Phe Thr Thr Asn Tyr Leu Lys

1 5 10 15

aaa gtt tta gca gga cgg gag gct acc cac gtc gat gaa agc ctt ttg 95

Lys Val Leu Ala Gly Arg Glu Ala Thr His Val Asp Glu Ser Leu Leu

20 25 30

gga gag gtg aag tac gca ttg gag ttt cca tgg cat tgc agt gtg cag 143

Gly Glu Val Lys Tyr Ala Leu Glu Phe Pro Trp His Cys Ser Val Gln

35 40 45

aga tgg gag gca agg agc ttt atc gaa ata ttt gga caa att gat tca 191

Arg Trp Glu Ala Arg Ser Phe Ile Glu Ile Phe Gly Gln Ile Asp Ser

50 55 60

gag ctt aag tcg aat ttg agc aaa aaa atg tta gag ttg gcg aaa ttg 239

Glu Leu Lys Ser Asn Leu Ser Lys Lys Met Leu Glu Leu Ala Lys Leu

65 70 75

gac ttc aat att ctg caa tgc aca cat cag aaa gaa ctg cag att atc 287

Asp Phe Asn Ile Leu Gln Cys Thr His Gln Lys Glu Leu Gln Ile Ile

80 85 90 95

tca agg tgg ttc gca gac tca agt ata gca tcc ctg aat ttc tat cgg 335

Ser Arg Trp Phe Ala Asp Ser Ser Ile Ala Ser Leu Asn Phe Tyr Arg

100 105 110

aaa tgt tac gtc gaa ttt tac ttt tgg atg gct gca gcc atc tcc gag 383

Lys Cys Tyr Val Glu Phe Tyr Phe Trp Met Ala Ala Ala Ile Ser Glu

115 120 125

ccg gag ttt tct gga agc aga gtt gcc ttc aca aaa att gct ata ctg 431

Pro Glu Phe Ser Gly Ser Arg Val Ala Phe Thr Lys Ile Ala Ile Leu

130 135 140

atg aca atg cta gat gac ctg tac gat act cac gga acc ttg gac caa 479

Met Thr Met Leu Asp Asp Leu Tyr Asp Thr His Gly Thr Leu Asp Gln

145 150 155

ctc aaa atc ttt aca gag gga gtg aga cga tgg gat gtt tcg ttg gta 527

Leu Lys Ile Phe Thr Glu Gly Val Arg Arg Trp Asp Val Ser Leu Val

160 165 170 175

gag ggc ctc cca gac ttc atg aaa att gca ttc gag ttc tgg tta aag 575

Glu Gly Leu Pro Asp Phe Met Lys Ile Ala Phe Glu Phe Trp Leu Lys

180 185 190

aca tct aat gaa ttg att gct gaa gct gtt aaa gcg caa ggg caa gat 623

Thr Ser Asn Glu Leu Ile Ala Glu Ala Val Lys Ala Gln Gly Gln Asp

195 200 205

atg gcg gcc tac ata aga aaa aat gca tgg gag cga tac ctt gaa gct 671

Met Ala Ala Tyr Ile Arg Lys Asn Ala Trp Glu Arg Tyr Leu Glu Ala

210 215 220

tat ctg caa gat gcg gaa tgg ata gcc act gga cat gtc ccc acc ttt 719

Tyr Leu Gln Asp Ala Glu Trp Ile Ala Thr Gly His Val Pro Thr Phe

225 230 235

gat gag tac ttg aat aat ggc aca cca aac act ggg atg tgt gta ttg 767

Asp Glu Tyr Leu Asn Asn Gly Thr Pro Asn Thr Gly Met Cys Val Leu

240 245 250 255

aat ttg att ccg ctt ctg tta atg ggt gaa cat tta cca atc gac att 815

Asn Leu Ile Pro Leu Leu Leu Met Gly Glu His Leu Pro Ile Asp Ile

260 265 270

ctg gag caa ata ttc ttg ccc tcc agg ttc cac cat ctc att gaa ttg 863

Leu Glu Gln Ile Phe Leu Pro Ser Arg Phe His His Leu Ile Glu Leu

275 280 285

gct tcc agg ctc gtc gat gac gcg aga gat ttc cag gcg gag aag gat 911

Ala Ser Arg Leu Val Asp Asp Ala Arg Asp Phe Gln Ala Glu Lys Asp

290 295 300

cat ggg gat tta tcg tgt att gag tgt tat tta aaa gat cat cct gag 959

His Gly Asp Leu Ser Cys Ile Glu Cys Tyr Leu Lys Asp His Pro Glu

305 310 315

tct aca gta gaa gat gct tta aat cat gtt aat ggc ctc ctt ggc aat 1007

Ser Thr Val Glu Asp Ala Leu Asn His Val Asn Gly Leu Leu Gly Asn

320 325 330 335

tgc ctt ctg gaa atg aat tgg aag ttc tta aag aag cag gac agt gtg 1055

Cys Leu Leu Glu Met Asn Trp Lys Phe Leu Lys Lys Gln Asp Ser Val

340 345 350

cca ctc tcg tgt aag aag tac agc ttc cat gta ttg gca cga agc atc 1103

Pro Leu Ser Cys Lys Lys Tyr Ser Phe His Val Leu Ala Arg Ser Ile

355 360 365

caa ttc atg tac aat caa ggc gat ggc ttc tcc att tcg aac aaa gtg 1151

Gln Phe Met Tyr Asn Gln Gly Asp Gly Phe Ser Ile Ser Asn Lys Val

370 375 380

atc aag gat caa gtg cag aaa gtt ctt att gtc ccc gtg cct att tga 1199

Ile Lys Asp Gln Val Gln Lys Val Leu Ile Val Pro Val Pro Ile

385 390 395

tagtagatac tagatagtag attagtagct attagtattt atttcatatc aatatttact 1259

aatgctgatg atggttaaag tccattcaga ccaatctttg gtttattgga cttaaataaa 1319

tgaattaatt agtttgtttt aaaattgtac tatttactgt tggaaataat gttttcatta 1379

ttgaaataac tagcacaact attttagtgt ggttgat 1416

<210> SEQ ID NO 20

<211> LENGTH: 398

<212> TYPE: PRT

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 20

Lys Val Met Glu Glu Ala Lys Ala Phe Thr Thr Asn Tyr Leu Lys Lys

1 5 10 15

Val Leu Ala Gly Arg Glu Ala Thr His Val Asp Glu Ser Leu Leu Gly

20 25 30

Glu Val Lys Tyr Ala Leu Glu Phe Pro Trp His Cys Ser Val Gln Arg

35 40 45

Trp Glu Ala Arg Ser Phe Ile Glu Ile Phe Gly Gln Ile Asp Ser Glu

50 55 60

Leu Lys Ser Asn Leu Ser Lys Lys Met Leu Glu Leu Ala Lys Leu Asp

65 70 75 80

Phe Asn Ile Leu Gln Cys Thr His Gln Lys Glu Leu Gln Ile Ile Ser

85 90 95

Arg Trp Phe Ala Asp Ser Ser Ile Ala Ser Leu Asn Phe Tyr Arg Lys

100 105 110

Cys Tyr Val Glu Phe Tyr Phe Trp Met Ala Ala Ala Ile Ser Glu Pro

115 120 125

Glu Phe Ser Gly Ser Arg Val Ala Phe Thr Lys Ile Ala Ile Leu Met

130 135 140

Thr Met Leu Asp Asp Leu Tyr Asp Thr His Gly Thr Leu Asp Gln Leu

145 150 155 160

Lys Ile Phe Thr Glu Gly Val Arg Arg Trp Asp Val Ser Leu Val Glu

165 170 175

Gly Leu Pro Asp Phe Met Lys Ile Ala Phe Glu Phe Trp Leu Lys Thr

180 185 190

Ser Asn Glu Leu Ile Ala Glu Ala Val Lys Ala Gln Gly Gln Asp Met

195 200 205

Ala Ala Tyr Ile Arg Lys Asn Ala Trp Glu Arg Tyr Leu Glu Ala Tyr

210 215 220

Leu Gln Asp Ala Glu Trp Ile Ala Thr Gly His Val Pro Thr Phe Asp

225 230 235 240

Glu Tyr Leu Asn Asn Gly Thr Pro Asn Thr Gly Met Cys Val Leu Asn

245 250 255

Leu Ile Pro Leu Leu Leu Met Gly Glu His Leu Pro Ile Asp Ile Leu

260 265 270

Glu Gln Ile Phe Leu Pro Ser Arg Phe His His Leu Ile Glu Leu Ala

275 280 285

Ser Arg Leu Val Asp Asp Ala Arg Asp Phe Gln Ala Glu Lys Asp His

290 295 300

Gly Asp Leu Ser Cys Ile Glu Cys Tyr Leu Lys Asp His Pro Glu Ser

305 310 315 320

Thr Val Glu Asp Ala Leu Asn His Val Asn Gly Leu Leu Gly Asn Cys

325 330 335

Leu Leu Glu Met Asn Trp Lys Phe Leu Lys Lys Gln Asp Ser Val Pro

340 345 350

Leu Ser Cys Lys Lys Tyr Ser Phe His Val Leu Ala Arg Ser Ile Gln

355 360 365

Phe Met Tyr Asn Gln Gly Asp Gly Phe Ser Ile Ser Asn Lys Val Ile

370 375 380

Lys Asp Gln Val Gln Lys Val Leu Ile Val Pro Val Pro Ile

385 390 395

<210> SEQ ID NO 21

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer E

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(23)

<223> OTHER INFORMATION: Wherein N=inosine residue

<400> SEQUENCE: 21

ggngaramrr tnatggarga rgc 23

<210> SEQ ID NO 22

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Degenerate Oligonucleotide Primer F

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(24)

<223> OTHER INFORMATION: Wherein N=inosine residue

<400> SEQUENCE: 22

garytncary tnhbnmgntg gtgg 24

<210> SEQ ID NO 23

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Degenerate Oligonucleotide Primer G

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(21)

<223> OTHER INFORMATION: Wherein N=inosine residue

<400> SEQUENCE: 23

ccarttnarn ccyttnacrt c 21

<210> SEQ ID NO 24

<211> LENGTH: 533

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 24

ggggaaaaaa tgatggagga agctgaaatc ttctctacca aatatttaaa agaagccctg 60

caaaagattc cggtctccag tctttcgcga gagatcgggg acgttttgga atatggttgg 120

cacacatatt tgccgcgatt ggaagcaagg aattacatcc aagtctttgg acaggacact 180

gagaacacga agtcatatgt gaagagcaaa aaacttttag aactcgcaaa attggagttc 240

aacatctttc aatccttact cgcatatccg cattgcaacc cattctgaca atggacatcc 300

cctttcctga tcatatcctc aaggaagttg acttcccatc aaagcttaac gacttggcat 360

gtgccatcct tcgattacga ggtgatacgc ggtgctacaa ggcggacagg gctcgtggag 420

aagaagcttc ctctatatca tgttatatga aagacaatcc tggagtatca gaggaagatg 480

ctctcgatca tatcaacgcc atgatcagtg acgaagtcaa aggcttcaat tgg 533

<210> SEQ ID NO 25

<211> LENGTH: 8

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Conserved Amino Acid Motif on Which the Sequence

of Primer D is based

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (3)..(3)

<223> OTHER INFORMATION: Wherein Xaa=THR or ILE

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (4)..(4)

<223> OTHER INFORMATION: Wherein Xaa=ILE or TYR or PHE

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (6)..(6)

<223> OTHER INFORMATION: Wherein Xaa=ALA or VAL

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (8)..(8)

<223> OTHER INFORMATION: Wherein Xaa=ALA or GLY

<400> SEQUENCE: 25

Asp Asp Xaa Xaa Asp Xaa Tyr Xaa

1 5

<210> SEQ ID NO 26

<211> LENGTH: 8

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Conserved Amino Acid Motif on Which the

Sequence of Primer E is based

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (3)..(3)

<223> OTHER INFORMATION: Wherein Xaa=LYS or THR

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (4)..(4)

<223> OTHER INFORMATION: Wherein Xaa=VAL or ILE

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (6)..(6)

<223> OTHER INFORMATION: Wherein Xaa=GLU or ASP

<400> SEQUENCE: 26

Gly Glu Xaa Xaa Met Xaa Glu Ala

1 5

<210> SEQ ID NO 27

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Conserved Amino Acid on Which the Sequence of

Primer F is based

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (2)..(2)

<223> OTHER INFORMATION: Wherein Xaa=PHE or TYR or ASP

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (3)..(3)

<223> OTHER INFORMATION: Wherein Xaa=ILE or LEU

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (4)..(4)

<223> OTHER INFORMATION: Wherein Xaa=THR or LEU or ARG

<400> SEQUENCE: 27

Gln Xaa Xaa Xaa Arg Trp Trp

1 5

<210> SEQ ID NO 28

<211> LENGTH: 8

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Conserved Amino Acid Motif on Which the

Sequence of Primer G is based

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (6)..(6)

<223> OTHER INFORMATION: Wherein Xaa=PHE or LEU

<400> SEQUENCE: 28

Asp Val Ile Lys Gly Xaa Asn Trp

1 5

<210> SEQ ID NO 29

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: T3 Primer Oligonucleotide

<400> SEQUENCE: 29

aattaaccct cactaaaggg 20

<210> SEQ ID NO 30

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: T7 Primer Oligonucleotide

<400> SEQUENCE: 30

gtaatacgac tcactatagg gc 22

<210> SEQ ID NO 31

<211> LENGTH: 2205

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (57)..(1943)

<223> OTHER INFORMATION:

<400> SEQUENCE: 31

gttatcttga gcttcctcca tataggccaa cacatatcat atcaaaggga gcaaga atg 59

Met

1

gct ctg gtt tct atc tca ccg ttg gct tcg aaa tct tgc ctg cgc aag 107

Ala Leu Val Ser Ile Ser Pro Leu Ala Ser Lys Ser Cys Leu Arg Lys

5 10 15

tcg ttg atc agt tca att cat gaa cat aag cct ccc tat aga aca atc 155

Ser Leu Ile Ser Ser Ile His Glu His Lys Pro Pro Tyr Arg Thr Ile

20 25 30

cca aat ctt gga atg cgt agg cga ggg aaa tct gtc acg cct tcc atg 203

Pro Asn Leu Gly Met Arg Arg Arg Gly Lys Ser Val Thr Pro Ser Met

35 40 45

agc atc agt ttg gcc acc gct gca cct gat gat ggt gta caa aga cgc 251

Ser Ile Ser Leu Ala Thr Ala Ala Pro Asp Asp Gly Val Gln Arg Arg

50 55 60 65

ata ggt gac tac cat tcc aat atc tgg gac gat gat ttc ata cag tct 299

Ile Gly Asp Tyr His Ser Asn Ile Trp Asp Asp Asp Phe Ile Gln Ser

70 75 80

cta tca acg cat tat ggg gaa ccc tct tac cag gaa cgt gct gag aga 347

Leu Ser Thr His Tyr Gly Glu Pro Ser Tyr Gln Glu Arg Ala Glu Arg

85 90 95

tta att gtg gag gta aag aag ata ttc aat tca atg tac ctg gat gat 395

Leu Ile Val Glu Val Lys Lys Ile Phe Asn Ser Met Tyr Leu Asp Asp

100 105 110

gga aga tta atg agt tcc ttt aat gat ctc atg caa cgc ctt tgg ata 443

Gly Arg Leu Met Ser Ser Phe Asn Asp Leu Met Gln Arg Leu Trp Ile

115 120 125

gtc gat agc gtt gaa cgt ttg ggg ata gct aga cat ttc aag aac gag 491

Val Asp Ser Val Glu Arg Leu Gly Ile Ala Arg His Phe Lys Asn Glu

130 135 140 145

ata aca tca gct ctg gat tat gtt ttc cgt tac tgg gag gaa aac ggc 539

Ile Thr Ser Ala Leu Asp Tyr Val Phe Arg Tyr Trp Glu Glu Asn Gly

150 155 160

att gga tgt ggg aga gac agt att gtt act gat ctc aac tca act gcg 587

Ile Gly Cys Gly Arg Asp Ser Ile Val Thr Asp Leu Asn Ser Thr Ala

165 170 175

ttg ggg ttt cga act ctt cga tta cac ggg tac act gta tct cca gag 635

Leu Gly Phe Arg Thr Leu Arg Leu His Gly Tyr Thr Val Ser Pro Glu

180 185 190

gtt tta aaa gct ttt caa gat caa aat gga cag ttt gta tgc tcc ccc 683

Val Leu Lys Ala Phe Gln Asp Gln Asn Gly Gln Phe Val Cys Ser Pro

195 200 205

ggt cag aca gag ggt gag atc aga agc gtt ctt aac tta tat cgg gct 731

Gly Gln Thr Glu Gly Glu Ile Arg Ser Val Leu Asn Leu Tyr Arg Ala

210 215 220 225

tcc ctc att gcc ttc cct ggt gag aaa gtt atg gaa gaa gct gaa atc 779

Ser Leu Ile Ala Phe Pro Gly Glu Lys Val Met Glu Glu Ala Glu Ile

230 235 240

ttc tcc aca aga tat ttg aaa gaa gct cta caa aag att cca gtc tcc 827

Phe Ser Thr Arg Tyr Leu Lys Glu Ala Leu Gln Lys Ile Pro Val Ser

245 250 255

gct ctt tca caa gag ata aag ttt gtt atg gaa tat ggc tgg cac aca 875

Ala Leu Ser Gln Glu Ile Lys Phe Val Met Glu Tyr Gly Trp His Thr

260 265 270

aat ttg cca aga ttg gaa gca aga aat tac ata gac aca ctt gag aaa 923

Asn Leu Pro Arg Leu Glu Ala Arg Asn Tyr Ile Asp Thr Leu Glu Lys

275 280 285

gac acc agt gca tgg ctc aat aaa aat gct ggg aag aag ctt tta gaa 971

Asp Thr Ser Ala Trp Leu Asn Lys Asn Ala Gly Lys Lys Leu Leu Glu

290 295 300 305

ctt gca aaa ttg gag ttc aat ata ttt aac tcc tta caa caa aag gaa 1019

Leu Ala Lys Leu Glu Phe Asn Ile Phe Asn Ser Leu Gln Gln Lys Glu

310 315 320

tta caa tat ctt ttg aga tgg tgg aaa gag tcg gat ttg cct aaa ttg 1067

Leu Gln Tyr Leu Leu Arg Trp Trp Lys Glu Ser Asp Leu Pro Lys Leu

325 330 335

aca ttt gct cgg cat cgt cat gtg gaa ttc tac act ttg gcc tct tgt 1115

Thr Phe Ala Arg His Arg His Val Glu Phe Tyr Thr Leu Ala Ser Cys

340 345 350

att gcc att gac cca aaa cat tct gca ttc aga cta ggc ttc gcc aaa 1163

Ile Ala Ile Asp Pro Lys His Ser Ala Phe Arg Leu Gly Phe Ala Lys

355 360 365

atg tgt cat ctt gtc aca gtt ttg gac gat att tac gac act ttt gga 1211

Met Cys His Leu Val Thr Val Leu Asp Asp Ile Tyr Asp Thr Phe Gly

370 375 380 385

acg att gac gag ctt gaa ctc ttc aca tct gca att aag aga tgg aat 1259

Thr Ile Asp Glu Leu Glu Leu Phe Thr Ser Ala Ile Lys Arg Trp Asn

390 395 400

tca tca gag ata gaa cac ctt cca gaa tat atg aaa tgt gtg tac atg 1307

Ser Ser Glu Ile Glu His Leu Pro Glu Tyr Met Lys Cys Val Tyr Met

405 410 415

gtc gtg ttt gaa act gta aat gaa ctg aca cga gag gcg gag aag act 1355

Val Val Phe Glu Thr Val Asn Glu Leu Thr Arg Glu Ala Glu Lys Thr

420 425 430

caa ggg aga aac act ctc aac tat gtt cga aag gct tgg gag gct tat 1403

Gln Gly Arg Asn Thr Leu Asn Tyr Val Arg Lys Ala Trp Glu Ala Tyr

435 440 445

ttt gat tca tat atg gaa gaa gca aaa tgg atc tct aat ggt tat ctg 1451

Phe Asp Ser Tyr Met Glu Glu Ala Lys Trp Ile Ser Asn Gly Tyr Leu

450 455 460 465

cca acg ttt gaa gag tac cat gag aat ggg aaa gtg agc tct gca tat 1499

Pro Thr Phe Glu Glu Tyr His Glu Asn Gly Lys Val Ser Ser Ala Tyr

470 475 480

cgc gta gca aca ttg caa ccc atc ctc act ttg aat gca tgg ctt cct 1547

Arg Val Ala Thr Leu Gln Pro Ile Leu Thr Leu Asn Ala Trp Leu Pro

485 490 495

gat tac atc ttg aag gga att gat ttt cca tcc agg ttc aat gat ttg 1595

Asp Tyr Ile Leu Lys Gly Ile Asp Phe Pro Ser Arg Phe Asn Asp Leu

500 505 510

gca tcg tcc ttc ctt cgg cta cga ggt gac aca cgc tgc tac aag gcc 1643

Ala Ser Ser Phe Leu Arg Leu Arg Gly Asp Thr Arg Cys Tyr Lys Ala

515 520 525

gat agg gat cgt ggt gaa gaa gct tcg tgt ata tca tgt tat atg aaa 1691

Asp Arg Asp Arg Gly Glu Glu Ala Ser Cys Ile Ser Cys Tyr Met Lys

530 535 540 545

gac aat cct gga tca acc gaa gaa gat gcc ctc aat cat atc aat gcc 1739

Asp Asn Pro Gly Ser Thr Glu Glu Asp Ala Leu Asn His Ile Asn Ala

550 555 560

atg gtc aat gac ata atc aaa gaa tta aat tgg gaa ctt cta aga tcc 1787

Met Val Asn Asp Ile Ile Lys Glu Leu Asn Trp Glu Leu Leu Arg Ser

565 570 575

aac gac aat att cca atg ctg gcc aag aaa cat gct ttt gac ata aca 1835

Asn Asp Asn Ile Pro Met Leu Ala Lys Lys His Ala Phe Asp Ile Thr

580 585 590

aga gct ctc cac cat ctc tac ata tat cga gat ggc ttt agt gtt gcc 1883

Arg Ala Leu His His Leu Tyr Ile Tyr Arg Asp Gly Phe Ser Val Ala

595 600 605

aac aag gaa aca aaa aaa ttg gtt atg gaa aca ctc ctt gaa tct atg 1931

Asn Lys Glu Thr Lys Lys Leu Val Met Glu Thr Leu Leu Glu Ser Met

610 615 620 625

ctt ttt taa cta taaccatatc cataataata agctcataat gctaaattat 1983

Leu Phe Leu

tggccttatg acatagttta tgtatgtact tgtgtgaatt caatcatatc gtgtgggtat 2043

gattaaaaag ctagagctta ctaggttagt aacatggtga taaaagttat aaaatgtgag 2103

ttatagagat acccatgttg aataatgaat tacaaaaaga gaaatttatg tagaataaga 2163

ttggaagctt ttcaattgtt ttaaaaaaaa aaaaaaaaaa aa 2205

<210> SEQ ID NO 32

<211> LENGTH: 627

<212> TYPE: PRT

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 32

Met Ala Leu Val Ser Ile Ser Pro Leu Ala Ser Lys Ser Cys Leu Arg

1 5 10 15

Lys Ser Leu Ile Ser Ser Ile His Glu His Lys Pro Pro Tyr Arg Thr

20 25 30

Ile Pro Asn Leu Gly Met Arg Arg Arg Gly Lys Ser Val Thr Pro Ser

35 40 45

Met Ser Ile Ser Leu Ala Thr Ala Ala Pro Asp Asp Gly Val Gln Arg

50 55 60

Arg Ile Gly Asp Tyr His Ser Asn Ile Trp Asp Asp Asp Phe Ile Gln

65 70 75 80

Ser Leu Ser Thr His Tyr Gly Glu Pro Ser Tyr Gln Glu Arg Ala Glu

85 90 95

Arg Leu Ile Val Glu Val Lys Lys Ile Phe Asn Ser Met Tyr Leu Asp

100 105 110

Asp Gly Arg Leu Met Ser Ser Phe Asn Asp Leu Met Gln Arg Leu Trp

115 120 125

Ile Val Asp Ser Val Glu Arg Leu Gly Ile Ala Arg His Phe Lys Asn

130 135 140

Glu Ile Thr Ser Ala Leu Asp Tyr Val Phe Arg Tyr Trp Glu Glu Asn

145 150 155 160

Gly Ile Gly Cys Gly Arg Asp Ser Ile Val Thr Asp Leu Asn Ser Thr

165 170 175

Ala Leu Gly Phe Arg Thr Leu Arg Leu His Gly Tyr Thr Val Ser Pro

180 185 190

Glu Val Leu Lys Ala Phe Gln Asp Gln Asn Gly Gln Phe Val Cys Ser

195 200 205

Pro Gly Gln Thr Glu Gly Glu Ile Arg Ser Val Leu Asn Leu Tyr Arg

210 215 220

Ala Ser Leu Ile Ala Phe Pro Gly Glu Lys Val Met Glu Glu Ala Glu

225 230 235 240

Ile Phe Ser Thr Arg Tyr Leu Lys Glu Ala Leu Gln Lys Ile Pro Val

245 250 255

Ser Ala Leu Ser Gln Glu Ile Lys Phe Val Met Glu Tyr Gly Trp His

260 265 270

Thr Asn Leu Pro Arg Leu Glu Ala Arg Asn Tyr Ile Asp Thr Leu Glu

275 280 285

Lys Asp Thr Ser Ala Trp Leu Asn Lys Asn Ala Gly Lys Lys Leu Leu

290 295 300

Glu Leu Ala Lys Leu Glu Phe Asn Ile Phe Asn Ser Leu Gln Gln Lys

305 310 315 320

Glu Leu Gln Tyr Leu Leu Arg Trp Trp Lys Glu Ser Asp Leu Pro Lys

325 330 335

Leu Thr Phe Ala Arg His Arg His Val Glu Phe Tyr Thr Leu Ala Ser

340 345 350

Cys Ile Ala Ile Asp Pro Lys His Ser Ala Phe Arg Leu Gly Phe Ala

355 360 365

Lys Met Cys His Leu Val Thr Val Leu Asp Asp Ile Tyr Asp Thr Phe

370 375 380

Gly Thr Ile Asp Glu Leu Glu Leu Phe Thr Ser Ala Ile Lys Arg Trp

385 390 395 400

Asn Ser Ser Glu Ile Glu His Leu Pro Glu Tyr Met Lys Cys Val Tyr

405 410 415

Met Val Val Phe Glu Thr Val Asn Glu Leu Thr Arg Glu Ala Glu Lys

420 425 430

Thr Gln Gly Arg Asn Thr Leu Asn Tyr Val Arg Lys Ala Trp Glu Ala

435 440 445

Tyr Phe Asp Ser Tyr Met Glu Glu Ala Lys Trp Ile Ser Asn Gly Tyr

450 455 460

Leu Pro Thr Phe Glu Glu Tyr His Glu Asn Gly Lys Val Ser Ser Ala

465 470 475 480

Tyr Arg Val Ala Thr Leu Gln Pro Ile Leu Thr Leu Asn Ala Trp Leu

485 490 495

Pro Asp Tyr Ile Leu Lys Gly Ile Asp Phe Pro Ser Arg Phe Asn Asp

500 505 510

Leu Ala Ser Ser Phe Leu Arg Leu Arg Gly Asp Thr Arg Cys Tyr Lys

515 520 525

Ala Asp Arg Asp Arg Gly Glu Glu Ala Ser Cys Ile Ser Cys Tyr Met

530 535 540

Lys Asp Asn Pro Gly Ser Thr Glu Glu Asp Ala Leu Asn His Ile Asn

545 550 555 560

Ala Met Val Asn Asp Ile Ile Lys Glu Leu Asn Trp Glu Leu Leu Arg

565 570 575

Ser Asn Asp Asn Ile Pro Met Leu Ala Lys Lys His Ala Phe Asp Ile

580 585 590

Thr Arg Ala Leu His His Leu Tyr Ile Tyr Arg Asp Gly Phe Ser Val

595 600 605

Ala Asn Lys Glu Thr Lys Lys Leu Val Met Glu Thr Leu Leu Glu Ser

610 615 620

Met Leu Phe

625

<210> SEQ ID NO 33

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Primer 2.2 BamHI

<400> SEQUENCE: 33

caaagggatc cagaatggct ctgg 24

<210> SEQ ID NO 34

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Primer 2.2 Not I

<400> SEQUENCE: 34

agtaagcggc cgctttttaa tcatacccac 30

<210> SEQ ID NO 35

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Primer 3.18 EcoRI

<400> SEQUENCE: 35

ctgcaggaat tcggcacgag c 21

<210> SEQ ID NO 36

<211> LENGTH: 27

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Primer3.18 SmaI

<400> SEQUENCE: 36

catagccccg ggcatagatt tgagctg 27

<210> SEQ ID NO 37

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Primer 10 NdeI

<400> SEQUENCE: 37

ggcaggaaca tatggctctc ctttctatcg 30

<210> SEQ ID NO 38

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Primer 10 BamHI

<400> SEQUENCE: 38

tctagaacta gtggatcccc cgggctgcag 30

<210> SEQ ID NO 39

<211> LENGTH: 18

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Primer JB29

<400> SEQUENCE: 39

ctaccattcc aatatctg 18

<210> SEQ ID NO 40

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Primer 2-8

<400> SEQUENCE: 40

gttggatctt agaagttccc 20

<210> SEQ ID NO 41

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Primer 3-9

<400> SEQUENCE: 41

tttccattcc aacctctggg 20

<210> SEQ ID NO 42

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Primer 3-11

<400> SEQUENCE: 42

cgtaatggaa agctctggcg 20

<210> SEQ ID NO 43

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Primer 7-1

<400> SEQUENCE: 43

ccttacacgc ctttggatgg 20

<210> SEQ ID NO 44

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: PCR Oligonucleotide Sequence 7-3

<400> SEQUENCE: 44

tctgttgatc caggatggtc 20

<210> SEQ ID NO 45

<211> LENGTH: 5

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Conserved Amino Acid Motif Common to All Prenyl

Transferases

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (3)..(4)

<223> OTHER INFORMATION: Wherein Xaa=any amino acid

<400> SEQUENCE: 45

Asp Asp Xaa Xaa Asp

1 5

<210> SEQ ID NO 46

<211> LENGTH: 8

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino Acid Motif from which Oligonucleotide

Primers can be Synthesized that Hybridize to the Monoterpene

Synthases of the Present Invention

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (4)..(4)

<223> OTHER INFORMATION: Wherein Xaa=LEU or ILE or VAL

<400> SEQUENCE: 46

His Ser Asn Xaa Thr Asp Asp Asp

1 5

<210> SEQ ID NO 47

<211> LENGTH: 6

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino Acid Motif from which Degenerate

Oligonucleotides can be Constructed that Hybridize to the

Monoterpene Synthases of the Present Invention

<400> SEQUENCE: 47

Ala Leu Asp Tyr Val Tyr

1 5

<210> SEQ ID NO 48

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino Acid Motif from which Degenerate

Oligonucleotide Sequences can be Constructed that Hybridize

to the Monoterpene Synthases of the Present Invention

<400> SEQUENCE: 48

Glu Leu Ala Lys Leu Glu Phe

1 5

<210> SEQ ID NO 49

<211> LENGTH: 6

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino Acid Motif from which Degenerate

Oligonucleotide Sequences an be Constructed that Hybridize

to the Monoterpene Synthase Clones of the Present Invention

<400> SEQUENCE: 49

Arg Trp Trp Lys Glu Ser

1 5

<210> SEQ ID NO 50

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino Acid Motif from which Oligonucleotide

Sequences can be Constructed that Hybridize to the Monoterpene

Synthase Clones of the Present Invention

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (1)..(1)

<223> OTHER INFORMATION: Wherein Xaa=VAL or ILE or LEU

<400> SEQUENCE: 50

Xaa Leu Asp Asp Met Tyr Asp

1 5

<210> SEQ ID NO 51

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino Acid Motif from which Oligonucleotide

Sequences can be Constructed that Hybridize to the Monoterpene

Synthase Clones of the Present Invention

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (1)..(1)

<223> OTHER INFORMATION: Wherein Xaa=VAL or ILE or LEU

<400> SEQUENCE: 51

Xaa Leu Asp Asp Leu Tyr Asp

1 5

<210> SEQ ID NO 52

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino Acid Motif from which Oligonucleotide

Sequences can be Constructed that Hybridize to the Monoterpene

Synthase Clones of the Present Invention

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (1)..(1)

<223> OTHER INFORMATION: Wherein Xaa=VAL or ILE or LEU

<400> SEQUENCE: 52

Xaa Leu Asp Asp Ile Tyr Asp

1 5

<210> SEQ ID NO 53

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Amino Acid Motif from which Oligonucleotide

Sequences can be Constructed that Hybridize to the Monoterpene

Synthase Clones of the Present Invention

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (6)..(6)

<223> OTHER INFORMATION: Wherein Xaa=ASN or HIS

<400> SEQUENCE: 53

Cys Tyr Met Lys Asp Xaa Pro

1 5

<210> SEQ ID NO 54

<211> LENGTH: 9

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Exemplary Oligonucleotide that Corresponds

to Peptide Sequence MetMetMet

<400> SEQUENCE: 54

atgatgatg 9

<210> SEQ ID NO 55

<211> LENGTH: 9

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Exemplary Oligonucleotide Sequence that

Corresponds to Peptide Sequence MetMetMet

<400> SEQUENCE: 55

tactactac 9

<210> SEQ ID NO 56

<211> LENGTH: 9

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Exemplary Oligonucleotide that Corresponds to

Peptide Sequence MetMetMet

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(9)

<223> OTHER INFORMATION: Wherein N=inosine

<400> SEQUENCE: 56

nacnacnac 9

<210> SEQ ID NO 57

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Corresponding to Amino Acid

Sequence Set Forth in SEQ ID NO:46

<400> SEQUENCE: 57

gtgtcgttgg agaccctgct gctg 24

<210> SEQ ID NO 58

<211> LENGTH: 18

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Corresponding to Amino Acid

Sequence Set Forth in SEQ ID NO:47

<400> SEQUENCE: 58

cgggagctga tgcagatg 18

<210> SEQ ID NO 59

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Corresponding to Conserved

Amino Acid Sequence Set Forth in SEQ ID NO:48

<400> SEQUENCE: 59

ctcgagcggt tcgagctcaa g 21

<210> SEQ ID NO 60

<211> LENGTH: 18

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Corresponding to Conserved

Amino Acid Sequence Set Forth in SEQ ID NO:49

<400> SEQUENCE: 60

gccaccacct tcctctcg 18

<210> SEQ ID NO 61

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Sequence Corresponding to

Amino Acid Sequence Set Forth in SEQ ID NO:50

<400> SEQUENCE: 61

gaggagctgc tgtacatgct g 21

<210> SEQ ID NO 62

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Corresponding to Conserved

Amino Acid Sequence Set Forth in SEQ ID NO:51

<400> SEQUENCE: 62

gaggagctgc tggagatgct g 21

<210> SEQ ID NO 63

<211> LENGTH: 293

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 63

cttaatgaat tggcgcaaga ggctgagaag actcaaggca gagatacgct caactatatt 60

cgcaatgctt atgagtctca ttttgattcg tttatgcacg aagcaaaatg gatctcaagt 120

ggttatctcc caacgtttga ggagtacttg aagaatggga aagttagttc cggttctcgc 180

acagccactt tacaacccat actcaccttg gatgtaccac ttcctaatta catactgcaa 240

gaaattgatt atccatctag gttcaatgac ttggcttcgt ccctccttcg cta 293

<210> SEQ ID NO 64

<211> LENGTH: 2013

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (36)..(1889)

<223> OTHER INFORMATION:

<400> SEQUENCE: 64

ttttgacgtg ccttcttatc tgatagcaag ctgaa atg gct ctt ctt tct att 53

Met Ala Leu Leu Ser Ile

1 5

act ccg ctg gtt tcc agg tcg tgc ctc agt tct tct cat gag att aag 101

Thr Pro Leu Val Ser Arg Ser Cys Leu Ser Ser Ser His Glu Ile Lys

10 15 20

gct ctc cgt aga aca atc cca act ctt gga atc tgc agg ccg ggg aaa 149

Ala Leu Arg Arg Thr Ile Pro Thr Leu Gly Ile Cys Arg Pro Gly Lys

25 30 35

tcc gtc gcg cat tcc ata aac atg tgt ttg aca agc gtc gca tct act 197

Ser Val Ala His Ser Ile Asn Met Cys Leu Thr Ser Val Ala Ser Thr

40 45 50

gat tct gta cag aga cgc gtg ggc aac tat cat tcc aac ctg tgg gac 245

Asp Ser Val Gln Arg Arg Val Gly Asn Tyr His Ser Asn Leu Trp Asp

55 60 65 70

gat gat ttc ata cag tct ctg atc tca acg cct tat gga gca cct gat 293

Asp Asp Phe Ile Gln Ser Leu Ile Ser Thr Pro Tyr Gly Ala Pro Asp

75 80 85

tac cgg gaa cgt gct gac aga ctt att ggg gaa gta aag gat ata atg 341

Tyr Arg Glu Arg Ala Asp Arg Leu Ile Gly Glu Val Lys Asp Ile Met

90 95 100

ttc aat ttc aag tcg ctg gaa gat gga ggc aat gat ctc ctt caa cga 389

Phe Asn Phe Lys Ser Leu Glu Asp Gly Gly Asn Asp Leu Leu Gln Arg

105 110 115

ctt ttg ctg gtc gat gac gtt gaa cgt ttg gga atc gac agg cat ttc 437

Leu Leu Leu Val Asp Asp Val Glu Arg Leu Gly Ile Asp Arg His Phe

120 125 130

aaa aaa gag ata aaa acg gca ctc gat tat gtt aac agt tat tgg aac 485

Lys Lys Glu Ile Lys Thr Ala Leu Asp Tyr Val Asn Ser Tyr Trp Asn

135 140 145 150

gaa aaa ggc att gga tgt ggg agg gag agt gtt gtg act gac ctc aac 533

Glu Lys Gly Ile Gly Cys Gly Arg Glu Ser Val Val Thr Asp Leu Asn

155 160 165

tca acc gcc ttg ggg ctt cga act ctc cga cta cac gga tac act gtg 581

Ser Thr Ala Leu Gly Leu Arg Thr Leu Arg Leu His Gly Tyr Thr Val

170 175 180

tct tca gat gtt ttg aac gtt ttt aaa gac aaa aat ggg caa ttt tcc 629

Ser Ser Asp Val Leu Asn Val Phe Lys Asp Lys Asn Gly Gln Phe Ser

185 190 195

tcc act gcc aat att cag ata gag gga gag att aga ggc gtt ctc aat 677

Ser Thr Ala Asn Ile Gln Ile Glu Gly Glu Ile Arg Gly Val Leu Asn

200 205 210

tta ttc agg gcc tcc ctc gtc gcc ttt ccc ggc gag aaa gtt atg gat 725

Leu Phe Arg Ala Ser Leu Val Ala Phe Pro Gly Glu Lys Val Met Asp

215 220 225 230

gaa gct gaa aca ttc tct aca aaa tat tta aga gaa gcc ctg caa aag 773

Glu Ala Glu Thr Phe Ser Thr Lys Tyr Leu Arg Glu Ala Leu Gln Lys

235 240 245

att ccg gca tcc agt ata ctt tca cta gag ata cgg gac gtt ctg gaa 821

Ile Pro Ala Ser Ser Ile Leu Ser Leu Glu Ile Arg Asp Val Leu Glu

250 255 260

tat ggt tgg cac acc aat ttg cca cgc ttg gaa gca agg aat tac atg 869

Tyr Gly Trp His Thr Asn Leu Pro Arg Leu Glu Ala Arg Asn Tyr Met

265 270 275

gac gtc ttt gga cag cac act aaa aat aag aac gcc gcc gag aaa ctt 917

Asp Val Phe Gly Gln His Thr Lys Asn Lys Asn Ala Ala Glu Lys Leu

280 285 290

tta gaa ctt gca aaa ttg gaa ttc aat ata ttt cac tcc tta caa gag 965

Leu Glu Leu Ala Lys Leu Glu Phe Asn Ile Phe His Ser Leu Gln Glu

295 300 305 310

aga gag tta aaa cat gtt tcc cga tgg tgg aaa gac tcg ggt tct cct 1013

Arg Glu Leu Lys His Val Ser Arg Trp Trp Lys Asp Ser Gly Ser Pro

315 320 325

gag atg acc ttc tgt cga cat cgt cac gtg gaa tac tac gct ttg gct 1061

Glu Met Thr Phe Cys Arg His Arg His Val Glu Tyr Tyr Ala Leu Ala

330 335 340

tcc tgc att gcg ttc gag cct caa cat tct gga ttc aga ctc ggc ttt 1109

Ser Cys Ile Ala Phe Glu Pro Gln His Ser Gly Phe Arg Leu Gly Phe

345 350 355

acc aag atg tct cat ctt atc acg gtt ctt gac gac atg tac gac gtc 1157

Thr Lys Met Ser His Leu Ile Thr Val Leu Asp Asp Met Tyr Asp Val

360 365 370

ttc ggc aca gta gac gag ctg gaa ctc ttc aca gcg aca att aag aga 1205

Phe Gly Thr Val Asp Glu Leu Glu Leu Phe Thr Ala Thr Ile Lys Arg

375 380 385 390

tgg gat ccg tcc gcg atg gaa tgc ctt cca gaa tat atg aaa gga gtg 1253

Trp Asp Pro Ser Ala Met Glu Cys Leu Pro Glu Tyr Met Lys Gly Val

395 400 405

tac atg atg gtt tat cac acc gta aat gaa atg gct cga gtg gca gag 1301

Tyr Met Met Val Tyr His Thr Val Asn Glu Met Ala Arg Val Ala Glu

410 415 420

aag gct caa ggc cga gac acg ctc aac tat gca aga cag gct tgg gag 1349

Lys Ala Gln Gly Arg Asp Thr Leu Asn Tyr Ala Arg Gln Ala Trp Glu

425 430 435

gcg tgt ttt gat tcg tat atg cag gaa gca aag tgg atc gcc act ggt 1397

Ala Cys Phe Asp Ser Tyr Met Gln Glu Ala Lys Trp Ile Ala Thr Gly

440 445 450

tat ctg ccc acg ttt gag gag tac ttg gag aac ggg aaa gtt agc tct 1445

Tyr Leu Pro Thr Phe Glu Glu Tyr Leu Glu Asn Gly Lys Val Ser Ser

455 460 465 470

gct cat cgc cca tgc gca ctg caa ccc att ctg acg ttg gac atc ccc 1493

Ala His Arg Pro Cys Ala Leu Gln Pro Ile Leu Thr Leu Asp Ile Pro

475 480 485

ttt cct gat cac atc ctc aag gaa gtt gac ttc cca tcg aag ctc aat 1541

Phe Pro Asp His Ile Leu Lys Glu Val Asp Phe Pro Ser Lys Leu Asn

490 495 500

gac ttg ata tgt atc atc ctt cga tta aga ggt gat aca cgg tgc tac 1589

Asp Leu Ile Cys Ile Ile Leu Arg Leu Arg Gly Asp Thr Arg Cys Tyr

505 510 515

aag gca gac agg gcc cgt gga gaa gaa gct tcg tct ata tca tgt tat 1637

Lys Ala Asp Arg Ala Arg Gly Glu Glu Ala Ser Ser Ile Ser Cys Tyr

520 525 530

atg aaa gac aat cct gga tta acg gaa gaa gat gct ctg aat cat atc 1685

Met Lys Asp Asn Pro Gly Leu Thr Glu Glu Asp Ala Leu Asn His Ile

535 540 545 550

aac ttc atg atc agg gac gca atc aga gaa tta aat tgg gag ctt cta 1733

Asn Phe Met Ile Arg Asp Ala Ile Arg Glu Leu Asn Trp Glu Leu Leu

555 560 565

aag cca gac aac agt gtt ccc atc act tcc aag aaa cac gca ttt gac 1781

Lys Pro Asp Asn Ser Val Pro Ile Thr Ser Lys Lys His Ala Phe Asp

570 575 580

ata agc aga gtt tgg cat cac ggt tac aga tac cga gat ggc tac agc 1829

Ile Ser Arg Val Trp His His Gly Tyr Arg Tyr Arg Asp Gly Tyr Ser

585 590 595

ttt gcc aac gtt gaa aca aag agt ttg gtg atg aga acc gtc att gaa 1877

Phe Ala Asn Val Glu Thr Lys Ser Leu Val Met Arg Thr Val Ile Glu

600 605 610

cct gtg cct ttg taacaacact tcaaatctac aatattaact gaggatgccc 1929

Pro Val Pro Leu

615

tatgggtgta tatagggcac acaaaaataa atatggttgt gttagtaaag ctgtaattta 1989

tgaaaaaaaa aaaaaaaaaa aaaa 2013

<210> SEQ ID NO 65

<211> LENGTH: 618

<212> TYPE: PRT

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 65

Met Ala Leu Leu Ser Ile Thr Pro Leu Val Ser Arg Ser Cys Leu Ser

1 5 10 15

Ser Ser His Glu Ile Lys Ala Leu Arg Arg Thr Ile Pro Thr Leu Gly

20 25 30

Ile Cys Arg Pro Gly Lys Ser Val Ala His Ser Ile Asn Met Cys Leu

35 40 45

Thr Ser Val Ala Ser Thr Asp Ser Val Gln Arg Arg Val Gly Asn Tyr

50 55 60

His Ser Asn Leu Trp Asp Asp Asp Phe Ile Gln Ser Leu Ile Ser Thr

65 70 75 80

Pro Tyr Gly Ala Pro Asp Tyr Arg Glu Arg Ala Asp Arg Leu Ile Gly

85 90 95

Glu Val Lys Asp Ile Met Phe Asn Phe Lys Ser Leu Glu Asp Gly Gly

100 105 110

Asn Asp Leu Leu Gln Arg Leu Leu Leu Val Asp Asp Val Glu Arg Leu

115 120 125

Gly Ile Asp Arg His Phe Lys Lys Glu Ile Lys Thr Ala Leu Asp Tyr

130 135 140

Val Asn Ser Tyr Trp Asn Glu Lys Gly Ile Gly Cys Gly Arg Glu Ser

145 150 155 160

Val Val Thr Asp Leu Asn Ser Thr Ala Leu Gly Leu Arg Thr Leu Arg

165 170 175

Leu His Gly Tyr Thr Val Ser Ser Asp Val Leu Asn Val Phe Lys Asp

180 185 190

Lys Asn Gly Gln Phe Ser Ser Thr Ala Asn Ile Gln Ile Glu Gly Glu

195 200 205

Ile Arg Gly Val Leu Asn Leu Phe Arg Ala Ser Leu Val Ala Phe Pro

210 215 220

Gly Glu Lys Val Met Asp Glu Ala Glu Thr Phe Ser Thr Lys Tyr Leu

225 230 235 240

Arg Glu Ala Leu Gln Lys Ile Pro Ala Ser Ser Ile Leu Ser Leu Glu

245 250 255

Ile Arg Asp Val Leu Glu Tyr Gly Trp His Thr Asn Leu Pro Arg Leu

260 265 270

Glu Ala Arg Asn Tyr Met Asp Val Phe Gly Gln His Thr Lys Asn Lys

275 280 285

Asn Ala Ala Glu Lys Leu Leu Glu Leu Ala Lys Leu Glu Phe Asn Ile

290 295 300

Phe His Ser Leu Gln Glu Arg Glu Leu Lys His Val Ser Arg Trp Trp

305 310 315 320

Lys Asp Ser Gly Ser Pro Glu Met Thr Phe Cys Arg His Arg His Val

325 330 335

Glu Tyr Tyr Ala Leu Ala Ser Cys Ile Ala Phe Glu Pro Gln His Ser

340 345 350

Gly Phe Arg Leu Gly Phe Thr Lys Met Ser His Leu Ile Thr Val Leu

355 360 365

Asp Asp Met Tyr Asp Val Phe Gly Thr Val Asp Glu Leu Glu Leu Phe

370 375 380

Thr Ala Thr Ile Lys Arg Trp Asp Pro Ser Ala Met Glu Cys Leu Pro

385 390 395 400

Glu Tyr Met Lys Gly Val Tyr Met Met Val Tyr His Thr Val Asn Glu

405 410 415

Met Ala Arg Val Ala Glu Lys Ala Gln Gly Arg Asp Thr Leu Asn Tyr

420 425 430

Ala Arg Gln Ala Trp Glu Ala Cys Phe Asp Ser Tyr Met Gln Glu Ala

435 440 445

Lys Trp Ile Ala Thr Gly Tyr Leu Pro Thr Phe Glu Glu Tyr Leu Glu

450 455 460

Asn Gly Lys Val Ser Ser Ala His Arg Pro Cys Ala Leu Gln Pro Ile

465 470 475 480

Leu Thr Leu Asp Ile Pro Phe Pro Asp His Ile Leu Lys Glu Val Asp

485 490 495

Phe Pro Ser Lys Leu Asn Asp Leu Ile Cys Ile Ile Leu Arg Leu Arg

500 505 510

Gly Asp Thr Arg Cys Tyr Lys Ala Asp Arg Ala Arg Gly Glu Glu Ala

515 520 525

Ser Ser Ile Ser Cys Tyr Met Lys Asp Asn Pro Gly Leu Thr Glu Glu

530 535 540

Asp Ala Leu Asn His Ile Asn Phe Met Ile Arg Asp Ala Ile Arg Glu

545 550 555 560

Leu Asn Trp Glu Leu Leu Lys Pro Asp Asn Ser Val Pro Ile Thr Ser

565 570 575

Lys Lys His Ala Phe Asp Ile Ser Arg Val Trp His His Gly Tyr Arg

580 585 590

Tyr Arg Asp Gly Tyr Ser Phe Ala Asn Val Glu Thr Lys Ser Leu Val

595 600 605

Met Arg Thr Val Ile Glu Pro Val Pro Leu

610 615

<210> SEQ ID NO 66

<211> LENGTH: 2186

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (34)..(1923)

<223> OTHER INFORMATION:

<400> SEQUENCE: 66

cccaaatcct atatccgtta taagcgagca gga atg gct ctg gtt tct tcc gca 54

Met Ala Leu Val Ser Ser Ala

1 5

ccc aaa tcc tgc ctg cac aaa tcg ttg atc agg tct act cat cat gag 102

Pro Lys Ser Cys Leu His Lys Ser Leu Ile Arg Ser Thr His His Glu

10 15 20

ctc aag cct ctg cgc aga acc atc cca act ctt gga atg tgt agg cga 150

Leu Lys Pro Leu Arg Arg Thr Ile Pro Thr Leu Gly Met Cys Arg Arg

25 30 35

ggg aaa tct ttc aca cct tct gtg agc atg agt ttg acc acc gct gta 198

Gly Lys Ser Phe Thr Pro Ser Val Ser Met Ser Leu Thr Thr Ala Val

40 45 50 55

tct gat gat ggt cta caa aga cgc ata ggt gac tat cat tcc aat ctc 246

Ser Asp Asp Gly Leu Gln Arg Arg Ile Gly Asp Tyr His Ser Asn Leu

60 65 70

tgg gac gac gat ttc ata cag tct cta tca acg cct tat ggg gag cct 294

Trp Asp Asp Asp Phe Ile Gln Ser Leu Ser Thr Pro Tyr Gly Glu Pro

75 80 85

tct tac cga gaa cgt gct gag aaa ctg att ggg gaa gtg aag gag atg 342

Ser Tyr Arg Glu Arg Ala Glu Lys Leu Ile Gly Glu Val Lys Glu Met

90 95 100

ttc aat tca atg cca tcg gaa gat gga gaa tca atg agt ccc ctc aat 390

Phe Asn Ser Met Pro Ser Glu Asp Gly Glu Ser Met Ser Pro Leu Asn

105 110 115

gat ctt att gaa cga ctt tgg atg gtc gat agc gtt gaa cgt ttg ggg 438

Asp Leu Ile Glu Arg Leu Trp Met Val Asp Ser Val Glu Arg Leu Gly

120 125 130 135

att gat aga cat ttc aaa aaa gag ata aaa tca gcc ctt gat tat gtt 486

Ile Asp Arg His Phe Lys Lys Glu Ile Lys Ser Ala Leu Asp Tyr Val

140 145 150

tac agt tat tgg aac gaa aaa ggt att gga tgc ggt aga gat agt gtt 534

Tyr Ser Tyr Trp Asn Glu Lys Gly Ile Gly Cys Gly Arg Asp Ser Val

155 160 165

ttt cct gat gtc aac tcg act gcc tcg ggg ttt cga act ctt cgc cta 582

Phe Pro Asp Val Asn Ser Thr Ala Ser Gly Phe Arg Thr Leu Arg Leu

170 175 180

cac gga tac agt gtc tct tca gag gtt ttg aaa gta ttt caa gac caa 630

His Gly Tyr Ser Val Ser Ser Glu Val Leu Lys Val Phe Gln Asp Gln

185 190 195

aat ggg cag ttt gca ttc tct cct agt aca aaa gag aga gac atc aga 678

Asn Gly Gln Phe Ala Phe Ser Pro Ser Thr Lys Glu Arg Asp Ile Arg

200 205 210 215

acc gtt ctg aat tta tat cgg gct tct ttc att gcc ttt cct ggg gag 726

Thr Val Leu Asn Leu Tyr Arg Ala Ser Phe Ile Ala Phe Pro Gly Glu

220 225 230

aaa gtt atg gaa gag gct gaa att ttc tct tca aga tat ttg aaa gaa 774

Lys Val Met Glu Glu Ala Glu Ile Phe Ser Ser Arg Tyr Leu Lys Glu

235 240 245

gcc gtg caa aag att ccg gtc tcc agt ctt tca caa gaa ata gac tac 822

Ala Val Gln Lys Ile Pro Val Ser Ser Leu Ser Gln Glu Ile Asp Tyr

250 255 260

act ttg gaa tat ggt tgg cac aca aat atg cca aga ttg gaa aca agg 870

Thr Leu Glu Tyr Gly Trp His Thr Asn Met Pro Arg Leu Glu Thr Arg

265 270 275

aat tac tta gat gta ttt gga cat cct acc agt cca tgg ctc aag aag 918

Asn Tyr Leu Asp Val Phe Gly His Pro Thr Ser Pro Trp Leu Lys Lys

280 285 290 295

aaa agg acg caa tat ctg gac agc gaa aag ctt tta gaa ctc gca aaa 966

Lys Arg Thr Gln Tyr Leu Asp Ser Glu Lys Leu Leu Glu Leu Ala Lys

300 305 310

ttg gag ttc aac atc ttt cac tcc ctt caa cag aag gag tta cag tat 1014

Leu Glu Phe Asn Ile Phe His Ser Leu Gln Gln Lys Glu Leu Gln Tyr

315 320 325

ctc tcc aga tgg tgg ata cat tcg ggt ttg cct gaa ctg acc ttt ggt 1062

Leu Ser Arg Trp Trp Ile His Ser Gly Leu Pro Glu Leu Thr Phe Gly

330 335 340

cgg cat cgt cac gtg gaa tac tac acc ctg agc tct tgc att gcg act 1110

Arg His Arg His Val Glu Tyr Tyr Thr Leu Ser Ser Cys Ile Ala Thr

345 350 355

gag ccc aaa cat tct gca ttc aga ttg ggc ttt gcc aaa acg tgt cat 1158

Glu Pro Lys His Ser Ala Phe Arg Leu Gly Phe Ala Lys Thr Cys His

360 365 370 375

ctt atc acg gtt ctg gac gat atc tac gac act ttc gga acg atg gat 1206

Leu Ile Thr Val Leu Asp Asp Ile Tyr Asp Thr Phe Gly Thr Met Asp

380 385 390

gaa atc gaa ctc ttc aac gag gca gtt agg aga tgg aat ccg tcg gag 1254

Glu Ile Glu Leu Phe Asn Glu Ala Val Arg Arg Trp Asn Pro Ser Glu

395 400 405

aaa gaa cgc ctc cca gaa tat atg aaa gaa atc tac atg gca ctc tac 1302

Lys Glu Arg Leu Pro Glu Tyr Met Lys Glu Ile Tyr Met Ala Leu Tyr

410 415 420

gaa gcc tta act gac atg gcg cga gag gca gag aag aca caa ggc cga 1350

Glu Ala Leu Thr Asp Met Ala Arg Glu Ala Glu Lys Thr Gln Gly Arg

425 430 435

gac acg ctc aat tat gct aga aag gct tgg gaa gtt tat ctt gat tcg 1398

Asp Thr Leu Asn Tyr Ala Arg Lys Ala Trp Glu Val Tyr Leu Asp Ser

440 445 450 455

tat aca caa gaa gca aag tgg atc gcc agc ggt tat ctg cca act ttc 1446

Tyr Thr Gln Glu Ala Lys Trp Ile Ala Ser Gly Tyr Leu Pro Thr Phe

460 465 470

gag gag tac tta gag aac gcg aag gtt agc tct ggt cat cgt gca gcg 1494

Glu Glu Tyr Leu Glu Asn Ala Lys Val Ser Ser Gly His Arg Ala Ala

475 480 485

gca ttg aca ccc ctc ctg aca ttg gac gta ccg ctt cct gat gac gtc 1542

Ala Leu Thr Pro Leu Leu Thr Leu Asp Val Pro Leu Pro Asp Asp Val

490 495 500

ttg aag gga ata gat ttt cca tcg aga ttt aat gat ttg gca tct tcc 1590

Leu Lys Gly Ile Asp Phe Pro Ser Arg Phe Asn Asp Leu Ala Ser Ser

505 510 515

ttc ctt aga cta aga ggt gac aca cga tgc tac aag gca gac agg gac 1638

Phe Leu Arg Leu Arg Gly Asp Thr Arg Cys Tyr Lys Ala Asp Arg Asp

520 525 530 535

cga gga gaa gaa gcg tca agc ata tcg tgt tac atg aaa gac aat ccc 1686

Arg Gly Glu Glu Ala Ser Ser Ile Ser Cys Tyr Met Lys Asp Asn Pro

540 545 550

gga tta aca gag gaa gat gct ctc aat cat atc aat gcc atg atc aac 1734

Gly Leu Thr Glu Glu Asp Ala Leu Asn His Ile Asn Ala Met Ile Asn

555 560 565

gac ata atc aaa gaa tta aat tgg gaa ctt ctc aaa ccc gat agc aat 1782

Asp Ile Ile Lys Glu Leu Asn Trp Glu Leu Leu Lys Pro Asp Ser Asn

570 575 580

att cca atg act gca cgg aaa cat gct tat gag ata acc aga gct ttc 1830

Ile Pro Met Thr Ala Arg Lys His Ala Tyr Glu Ile Thr Arg Ala Phe

585 590 595

cac caa ctt tac aaa tat aga gat ggc ttc agc gtt gcc act caa gaa 1878

His Gln Leu Tyr Lys Tyr Arg Asp Gly Phe Ser Val Ala Thr Gln Glu

600 605 610 615

acg aaa agt ttg gtg agg aga acg gtc ctt gaa cca gtg cct ctt 1923

Thr Lys Ser Leu Val Arg Arg Thr Val Leu Glu Pro Val Pro Leu

620 625 630

taacaattta aaccttctat aataaattgg tgtaggctcc gctatgcgtt tatgcatgtg 1983

catgtctctc tatgtaacta gttgtatgcg tggtatgatt ataaaattgg aggttactcg 2043

gtcctcacat ggtaatatgt gagttgtgaa attctcaaaa aaaaaaaaaa aaaaaaaaaa 2103

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2163

aaaaaaaaaa aaaaaaaaaa aaa 2186

<210> SEQ ID NO 67

<211> LENGTH: 630

<212> TYPE: PRT

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 67

Met Ala Leu Val Ser Ser Ala Pro Lys Ser Cys Leu His Lys Ser Leu

1 5 10 15

Ile Arg Ser Thr His His Glu Leu Lys Pro Leu Arg Arg Thr Ile Pro

20 25 30

Thr Leu Gly Met Cys Arg Arg Gly Lys Ser Phe Thr Pro Ser Val Ser

35 40 45

Met Ser Leu Thr Thr Ala Val Ser Asp Asp Gly Leu Gln Arg Arg Ile

50 55 60

Gly Asp Tyr His Ser Asn Leu Trp Asp Asp Asp Phe Ile Gln Ser Leu

65 70 75 80

Ser Thr Pro Tyr Gly Glu Pro Ser Tyr Arg Glu Arg Ala Glu Lys Leu

85 90 95

Ile Gly Glu Val Lys Glu Met Phe Asn Ser Met Pro Ser Glu Asp Gly

100 105 110

Glu Ser Met Ser Pro Leu Asn Asp Leu Ile Glu Arg Leu Trp Met Val

115 120 125

Asp Ser Val Glu Arg Leu Gly Ile Asp Arg His Phe Lys Lys Glu Ile

130 135 140

Lys Ser Ala Leu Asp Tyr Val Tyr Ser Tyr Trp Asn Glu Lys Gly Ile

145 150 155 160

Gly Cys Gly Arg Asp Ser Val Phe Pro Asp Val Asn Ser Thr Ala Ser

165 170 175

Gly Phe Arg Thr Leu Arg Leu His Gly Tyr Ser Val Ser Ser Glu Val

180 185 190

Leu Lys Val Phe Gln Asp Gln Asn Gly Gln Phe Ala Phe Ser Pro Ser

195 200 205

Thr Lys Glu Arg Asp Ile Arg Thr Val Leu Asn Leu Tyr Arg Ala Ser

210 215 220

Phe Ile Ala Phe Pro Gly Glu Lys Val Met Glu Glu Ala Glu Ile Phe

225 230 235 240

Ser Ser Arg Tyr Leu Lys Glu Ala Val Gln Lys Ile Pro Val Ser Ser

245 250 255

Leu Ser Gln Glu Ile Asp Tyr Thr Leu Glu Tyr Gly Trp His Thr Asn

260 265 270

Met Pro Arg Leu Glu Thr Arg Asn Tyr Leu Asp Val Phe Gly His Pro

275 280 285

Thr Ser Pro Trp Leu Lys Lys Lys Arg Thr Gln Tyr Leu Asp Ser Glu

290 295 300

Lys Leu Leu Glu Leu Ala Lys Leu Glu Phe Asn Ile Phe His Ser Leu

305 310 315 320

Gln Gln Lys Glu Leu Gln Tyr Leu Ser Arg Trp Trp Ile His Ser Gly

325 330 335

Leu Pro Glu Leu Thr Phe Gly Arg His Arg His Val Glu Tyr Tyr Thr

340 345 350

Leu Ser Ser Cys Ile Ala Thr Glu Pro Lys His Ser Ala Phe Arg Leu

355 360 365

Gly Phe Ala Lys Thr Cys His Leu Ile Thr Val Leu Asp Asp Ile Tyr

370 375 380

Asp Thr Phe Gly Thr Met Asp Glu Ile Glu Leu Phe Asn Glu Ala Val

385 390 395 400

Arg Arg Trp Asn Pro Ser Glu Lys Glu Arg Leu Pro Glu Tyr Met Lys

405 410 415

Glu Ile Tyr Met Ala Leu Tyr Glu Ala Leu Thr Asp Met Ala Arg Glu

420 425 430

Ala Glu Lys Thr Gln Gly Arg Asp Thr Leu Asn Tyr Ala Arg Lys Ala

435 440 445

Trp Glu Val Tyr Leu Asp Ser Tyr Thr Gln Glu Ala Lys Trp Ile Ala

450 455 460

Ser Gly Tyr Leu Pro Thr Phe Glu Glu Tyr Leu Glu Asn Ala Lys Val

465 470 475 480

Ser Ser Gly His Arg Ala Ala Ala Leu Thr Pro Leu Leu Thr Leu Asp

485 490 495

Val Pro Leu Pro Asp Asp Val Leu Lys Gly Ile Asp Phe Pro Ser Arg

500 505 510

Phe Asn Asp Leu Ala Ser Ser Phe Leu Arg Leu Arg Gly Asp Thr Arg

515 520 525

Cys Tyr Lys Ala Asp Arg Asp Arg Gly Glu Glu Ala Ser Ser Ile Ser

530 535 540

Cys Tyr Met Lys Asp Asn Pro Gly Leu Thr Glu Glu Asp Ala Leu Asn

545 550 555 560

His Ile Asn Ala Met Ile Asn Asp Ile Ile Lys Glu Leu Asn Trp Glu

565 570 575

Leu Leu Lys Pro Asp Ser Asn Ile Pro Met Thr Ala Arg Lys His Ala

580 585 590

Tyr Glu Ile Thr Arg Ala Phe His Gln Leu Tyr Lys Tyr Arg Asp Gly

595 600 605

Phe Ser Val Ala Thr Gln Glu Thr Lys Ser Leu Val Arg Arg Thr Val

610 615 620

Leu Glu Pro Val Pro Leu

625 630

<210> SEQ ID NO 68

<211> LENGTH: 2429

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (35)..(1945)

<223> OTHER INFORMATION:

<400> SEQUENCE: 68

attaaagaag ctaccatagt ttaggcagga atgc atg gct ctc ctt tct atc gta 55

Met Ala Leu Leu Ser Ile Val

1 5

tct ttg cag gtt ccc aaa tcc tgc ggg ctg aaa tcg ttg atc agt tcc 103

Ser Leu Gln Val Pro Lys Ser Cys Gly Leu Lys Ser Leu Ile Ser Ser

10 15 20

agc aat gtg cag aag gct ctc tgt atc tct aca gca gtc cca act ctc 151

Ser Asn Val Gln Lys Ala Leu Cys Ile Ser Thr Ala Val Pro Thr Leu

25 30 35

aga atg cgt agg cga cag aaa gct ctg gtc atc aac atg aaa ttg acc 199

Arg Met Arg Arg Arg Gln Lys Ala Leu Val Ile Asn Met Lys Leu Thr

40 45 50 55

act gta tcc cat cgt gat gat aat ggt ggt ggt gta ctg caa aga cgc 247

Thr Val Ser His Arg Asp Asp Asn Gly Gly Gly Val Leu Gln Arg Arg

60 65 70

ata gcc gat cat cat ccc aac ctg tgg gaa gat gat ttc ata caa tca 295

Ile Ala Asp His His Pro Asn Leu Trp Glu Asp Asp Phe Ile Gln Ser

75 80 85

ttg tcc tca cct tat ggg gga tct tcg tac agt gaa cgt gct gtg aca 343

Leu Ser Ser Pro Tyr Gly Gly Ser Ser Tyr Ser Glu Arg Ala Val Thr

90 95 100

gtg gtt gag gaa gta aaa gag atg ttc aat tca ata cca aat aat aga 391

Val Val Glu Glu Val Lys Glu Met Phe Asn Ser Ile Pro Asn Asn Arg

105 110 115

gaa tta ttt ggt tcc caa aat gat ctc ctt aca cgc ctt tgg atg gtg 439

Glu Leu Phe Gly Ser Gln Asn Asp Leu Leu Thr Arg Leu Trp Met Val

120 125 130 135

gat agc att gaa cgt ctg ggg ata gat aga cat ttc caa aat gag ata 487

Asp Ser Ile Glu Arg Leu Gly Ile Asp Arg His Phe Gln Asn Glu Ile

140 145 150

aga gta gcc ctc gat tat gtt tac agt tat tgg aag gaa aag gaa ggc 535

Arg Val Ala Leu Asp Tyr Val Tyr Ser Tyr Trp Lys Glu Lys Glu Gly

155 160 165

att ggg tgt ggc aga gat tct act ttt cct gat ctc aac tcg act gct 583

Ile Gly Cys Gly Arg Asp Ser Thr Phe Pro Asp Leu Asn Ser Thr Ala

170 175 180

ctg gcg ctt cga act ctt cga ctg cac gga tac aat gtg tct tca gat 631

Leu Ala Leu Arg Thr Leu Arg Leu His Gly Tyr Asn Val Ser Ser Asp

185 190 195

gtg ctg gaa tac ttc aaa gat caa aag ggg cat ttt gcc tgc cct gca 679

Val Leu Glu Tyr Phe Lys Asp Gln Lys Gly His Phe Ala Cys Pro Ala

200 205 210 215

atc cta acc gag gga cag atc act aga agt gtt cta aat tta tat cgg 727

Ile Leu Thr Glu Gly Gln Ile Thr Arg Ser Val Leu Asn Leu Tyr Arg

220 225 230

gct tcc ctg gtc gcc ttt ccg ggg gag aaa gtt atg gaa gag gct gaa 775

Ala Ser Leu Val Ala Phe Pro Gly Glu Lys Val Met Glu Glu Ala Glu

235 240 245

atc ttc tcg gca tct tat ttg aaa gaa gtc tta caa aag att cca gtc 823

Ile Phe Ser Ala Ser Tyr Leu Lys Glu Val Leu Gln Lys Ile Pro Val

250 255 260

tcc agt ttt tca cga gag ata gaa tac gtt ttg gaa tat ggt tgg cac 871

Ser Ser Phe Ser Arg Glu Ile Glu Tyr Val Leu Glu Tyr Gly Trp His

265 270 275

aca aat ttg cca aga ttg gaa gca aga aat tat atc gac gtc tac ggg 919

Thr Asn Leu Pro Arg Leu Glu Ala Arg Asn Tyr Ile Asp Val Tyr Gly

280 285 290 295

cag gac agc tat gaa agt tca aac gag atg cca tat gtg aat acg cag 967

Gln Asp Ser Tyr Glu Ser Ser Asn Glu Met Pro Tyr Val Asn Thr Gln

300 305 310

aag ctt tta aaa ctt gca aaa ttg gag ttt aat atc ttt cac tct ttg 1015

Lys Leu Leu Lys Leu Ala Lys Leu Glu Phe Asn Ile Phe His Ser Leu

315 320 325

caa cag aaa gag ttg caa tat atc tct aga tgg tgg aaa gat tcg tgt 1063

Gln Gln Lys Glu Leu Gln Tyr Ile Ser Arg Trp Trp Lys Asp Ser Cys

330 335 340

tca tct cat ctg act ttt act cga cac cgt cac gtg gaa tac tac aca 1111

Ser Ser His Leu Thr Phe Thr Arg His Arg His Val Glu Tyr Tyr Thr

345 350 355

atg gca tct tgc att tct atg gag ccg aaa cac tcc gct ttc aga ttg 1159

Met Ala Ser Cys Ile Ser Met Glu Pro Lys His Ser Ala Phe Arg Leu

360 365 370 375

ggg ttt gtc aaa aca tgt cat ctt cta aca gtt ctg gat gat atg tat 1207

Gly Phe Val Lys Thr Cys His Leu Leu Thr Val Leu Asp Asp Met Tyr

380 385 390

gac act ttt gga aca ctg gac gaa ctc caa ctt ttt acg act gcc ttt 1255

Asp Thr Phe Gly Thr Leu Asp Glu Leu Gln Leu Phe Thr Thr Ala Phe

395 400 405

aag aga tgg gat ttg tca gag aca aag tgt ctt cca gaa tat atg aaa 1303

Lys Arg Trp Asp Leu Ser Glu Thr Lys Cys Leu Pro Glu Tyr Met Lys

410 415 420

gca gtg tac atg gac ttg tat caa tgt ctt aat gaa ttg gcg caa gag 1351

Ala Val Tyr Met Asp Leu Tyr Gln Cys Leu Asn Glu Leu Ala Gln Glu

425 430 435

gct gag aag act caa ggc aga gat acg ctc aac tat att cgc aat gct 1399

Ala Glu Lys Thr Gln Gly Arg Asp Thr Leu Asn Tyr Ile Arg Asn Ala

440 445 450 455

tat gag tct cat ttt gat tcg ttt atg cac gaa gca aaa tgg atc tca 1447

Tyr Glu Ser His Phe Asp Ser Phe Met His Glu Ala Lys Trp Ile Ser

460 465 470

agt ggt tat ctc cca acg ttt gag gag tac ttg aag aat ggg aaa gtt 1495

Ser Gly Tyr Leu Pro Thr Phe Glu Glu Tyr Leu Lys Asn Gly Lys Val

475 480 485

agt tcc ggt tct cgc aca gcc act tta caa ccc ata ctc acc ttg gat 1543

Ser Ser Gly Ser Arg Thr Ala Thr Leu Gln Pro Ile Leu Thr Leu Asp

490 495 500

gta cca ctt cct aat tac ata ctg caa gaa att gat tat cca tct agg 1591

Val Pro Leu Pro Asn Tyr Ile Leu Gln Glu Ile Asp Tyr Pro Ser Arg

505 510 515

ttc aat gac ttg gct tcg tcc ctc ctt cgg cta cgt ggt gac acg cgc 1639

Phe Asn Asp Leu Ala Ser Ser Leu Leu Arg Leu Arg Gly Asp Thr Arg

520 525 530 535

tgc tac aag gcg gat agg gct cgt gga gaa gaa gct tca gct ata tcg 1687

Cys Tyr Lys Ala Asp Arg Ala Arg Gly Glu Glu Ala Ser Ala Ile Ser

540 545 550

tgt tat atg aaa gac cat cct gga tca aca gag gaa gat gct ctc aat 1735

Cys Tyr Met Lys Asp His Pro Gly Ser Thr Glu Glu Asp Ala Leu Asn

555 560 565

cat atc aac gtc atg atc agt gat gca atc aga gaa tta aat tgg gag 1783

His Ile Asn Val Met Ile Ser Asp Ala Ile Arg Glu Leu Asn Trp Glu

570 575 580

ctt ctc aga cca gat agc aaa agt ccc atc tct tcc aag aaa cat gct 1831

Leu Leu Arg Pro Asp Ser Lys Ser Pro Ile Ser Ser Lys Lys His Ala

585 590 595

ttt gac atc acc aga gct ttc cat cac ctc tac aag tac cga gat ggt 1879

Phe Asp Ile Thr Arg Ala Phe His His Leu Tyr Lys Tyr Arg Asp Gly

600 605 610 615

tac act gtt gcg agt agt gaa aca aag aat ttg gtg atg aaa aca gtt 1927

Tyr Thr Val Ala Ser Ser Glu Thr Lys Asn Leu Val Met Lys Thr Val

620 625 630

ctt gaa cct gtg gca ttg taaaaaaata tcaaccgcat caaaatgcac 1975

Leu Glu Pro Val Ala Leu

635

ggagtttgta atttaatgca cttctcttat aatacacttc tctttagacc tgtagtgaag 2035

ccgatgcacc attacagtgt atatgggagc cagtctagtc tcaaaaagtt tgtaaatgtt 2095

attctatgat atactcttta gaccaaaagc tagatgccca tgaaaagcaa gtgttttaga 2155

attgcttctg gatttgctta aattttctcc atgattcttt agaaatgttg catccccaaa 2215

cttcactgcc atataagata acgggagtga caaggatttt aaagaggatt tttttttatg 2275

tcccgcatca caaggtttgt cgatttacag ttgttttcaa gactgaagta ggatttccac 2335

cctccattaa tcctcttctc gatgttatag tttcacttga gcttgtgatg gaagtcaatt 2395

cctagatatt tataagaaaa aaaaaaaaaa aaaa 2429

<210> SEQ ID NO 69

<211> LENGTH: 637

<212> TYPE: PRT

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 69

Met Ala Leu Leu Ser Ile Val Ser Leu Gln Val Pro Lys Ser Cys Gly

1 5 10 15

Leu Lys Ser Leu Ile Ser Ser Ser Asn Val Gln Lys Ala Leu Cys Ile

20 25 30

Ser Thr Ala Val Pro Thr Leu Arg Met Arg Arg Arg Gln Lys Ala Leu

35 40 45

Val Ile Asn Met Lys Leu Thr Thr Val Ser His Arg Asp Asp Asn Gly

50 55 60

Gly Gly Val Leu Gln Arg Arg Ile Ala Asp His His Pro Asn Leu Trp

65 70 75 80

Glu Asp Asp Phe Ile Gln Ser Leu Ser Ser Pro Tyr Gly Gly Ser Ser

85 90 95

Tyr Ser Glu Arg Ala Val Thr Val Val Glu Glu Val Lys Glu Met Phe

100 105 110

Asn Ser Ile Pro Asn Asn Arg Glu Leu Phe Gly Ser Gln Asn Asp Leu

115 120 125

Leu Thr Arg Leu Trp Met Val Asp Ser Ile Glu Arg Leu Gly Ile Asp

130 135 140

Arg His Phe Gln Asn Glu Ile Arg Val Ala Leu Asp Tyr Val Tyr Ser

145 150 155 160

Tyr Trp Lys Glu Lys Glu Gly Ile Gly Cys Gly Arg Asp Ser Thr Phe

165 170 175

Pro Asp Leu Asn Ser Thr Ala Leu Ala Leu Arg Thr Leu Arg Leu His

180 185 190

Gly Tyr Asn Val Ser Ser Asp Val Leu Glu Tyr Phe Lys Asp Gln Lys

195 200 205

Gly His Phe Ala Cys Pro Ala Ile Leu Thr Glu Gly Gln Ile Thr Arg

210 215 220

Ser Val Leu Asn Leu Tyr Arg Ala Ser Leu Val Ala Phe Pro Gly Glu

225 230 235 240

Lys Val Met Glu Glu Ala Glu Ile Phe Ser Ala Ser Tyr Leu Lys Glu

245 250 255

Val Leu Gln Lys Ile Pro Val Ser Ser Phe Ser Arg Glu Ile Glu Tyr

260 265 270

Val Leu Glu Tyr Gly Trp His Thr Asn Leu Pro Arg Leu Glu Ala Arg

275 280 285

Asn Tyr Ile Asp Val Tyr Gly Gln Asp Ser Tyr Glu Ser Ser Asn Glu

290 295 300

Met Pro Tyr Val Asn Thr Gln Lys Leu Leu Lys Leu Ala Lys Leu Glu

305 310 315 320

Phe Asn Ile Phe His Ser Leu Gln Gln Lys Glu Leu Gln Tyr Ile Ser

325 330 335

Arg Trp Trp Lys Asp Ser Cys Ser Ser His Leu Thr Phe Thr Arg His

340 345 350

Arg His Val Glu Tyr Tyr Thr Met Ala Ser Cys Ile Ser Met Glu Pro

355 360 365

Lys His Ser Ala Phe Arg Leu Gly Phe Val Lys Thr Cys His Leu Leu

370 375 380

Thr Val Leu Asp Asp Met Tyr Asp Thr Phe Gly Thr Leu Asp Glu Leu

385 390 395 400

Gln Leu Phe Thr Thr Ala Phe Lys Arg Trp Asp Leu Ser Glu Thr Lys

405 410 415

Cys Leu Pro Glu Tyr Met Lys Ala Val Tyr Met Asp Leu Tyr Gln Cys

420 425 430

Leu Asn Glu Leu Ala Gln Glu Ala Glu Lys Thr Gln Gly Arg Asp Thr

435 440 445

Leu Asn Tyr Ile Arg Asn Ala Tyr Glu Ser His Phe Asp Ser Phe Met

450 455 460

His Glu Ala Lys Trp Ile Ser Ser Gly Tyr Leu Pro Thr Phe Glu Glu

465 470 475 480

Tyr Leu Lys Asn Gly Lys Val Ser Ser Gly Ser Arg Thr Ala Thr Leu

485 490 495

Gln Pro Ile Leu Thr Leu Asp Val Pro Leu Pro Asn Tyr Ile Leu Gln

500 505 510

Glu Ile Asp Tyr Pro Ser Arg Phe Asn Asp Leu Ala Ser Ser Leu Leu

515 520 525

Arg Leu Arg Gly Asp Thr Arg Cys Tyr Lys Ala Asp Arg Ala Arg Gly

530 535 540

Glu Glu Ala Ser Ala Ile Ser Cys Tyr Met Lys Asp His Pro Gly Ser

545 550 555 560

Thr Glu Glu Asp Ala Leu Asn His Ile Asn Val Met Ile Ser Asp Ala

565 570 575

Ile Arg Glu Leu Asn Trp Glu Leu Leu Arg Pro Asp Ser Lys Ser Pro

580 585 590

Ile Ser Ser Lys Lys His Ala Phe Asp Ile Thr Arg Ala Phe His His

595 600 605

Leu Tyr Lys Tyr Arg Asp Gly Tyr Thr Val Ala Ser Ser Glu Thr Lys

610 615 620

Asn Leu Val Met Lys Thr Val Leu Glu Pro Val Ala Leu

625 630 635

<210> SEQ ID NO 70

<211> LENGTH: 696

<212> TYPE: DNA

<213> ORGANISM: Abies grandis

<400> SEQUENCE: 70

gcatttaaga gatgggatcc gtctgccaca gatttgcttc cagagtatat gaaagggttg 60

tacatggtgg tttacgaaac cgtaaatgaa attgctcgag aggcagacaa gtctcaaggc 120

cgagagacgc tcaacgatgc tcgacgagct tgggaggcct atcttgattc gtatatgaaa 180

gaagctgagt ggatctccag tggttatctg ccaacgtttg aggagtacat ggagaccagc 240

aaagttagtt ttggttatcg catattcgca ttgcaaccca tcctcactat ggatgttccc 300

cttactcacc acatcctgca ggaaatagac tttccattga ggtttaatga cttaatatgt 360

tccatccttc gacttaaaaa tgacactcgc tgctacaagg cggacagggc ccgtggagaa 420

gaagcttcgt gtatatcgtg ttatatgaaa gagaatcctg gatcaacaga ggaagatgct 480

atcaatcata tcaacgctat ggtcaataac ttaatcaaag aagtgaattg ggagcttctc 540

cgacaggacg gcaccgctca tattgcttgc aagaaacacg cttttgacat cctcaaaggt 600

tcccttcacg gctacaaata ccgagatggg ttcagcgttg ccaacaagga aaccaagaat 660

tgggtgagga gaacagtcct tgagtctgtg cctttg 696

<210> SEQ ID NO 71

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Reverse RACE Primer 10-2

<400> SEQUENCE: 71

acgaagcttc ttctccacgg 20

<210> SEQ ID NO 72

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Reverse RACE Primer 10-4

<400> SEQUENCE: 72

ggatcccatc tcttaactgc 20

<210> SEQ ID NO 73

<211> LENGTH: 27

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer AP1

<400> SEQUENCE: 73

ccatcctaat acgactcact atagggc 27

<210> SEQ ID NO 74

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer AP2

<400> SEQUENCE: 74

actcactata gggctcgagc ggc 23

<210> SEQ ID NO 75

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer AG9F

<400> SEQUENCE: 75

atggctcttg tttctatctt gccc 24

<210> SEQ ID NO 76

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer AG9R

<400> SEQUENCE: 76

ttacaaaggc acagactcaa ggac 24

<210> SEQ ID NO 77

<211> LENGTH: 1890

<212> TYPE: DNA

<213> ORGANISM: Abies Grandis

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (1)..(1890)

<223> OTHER INFORMATION:

<400> SEQUENCE: 77

atg gct ctt gtt tct atc ttg ccc ttg tct tcc aaa tcg gtc ctg cac 48

Met Ala Leu Val Ser Ile Leu Pro Leu Ser Ser Lys Ser Val Leu His

1 5 10 15

aaa tcg tgg atc gtt tct act tat gag cat aag gct atc agt aga aca 96

Lys Ser Trp Ile Val Ser Thr Tyr Glu His Lys Ala Ile Ser Arg Thr

20 25 30

atc cca aat ctt gga ttg cgt ggg cga ggg aaa tct gtg aca cat tcc 144

Ile Pro Asn Leu Gly Leu Arg Gly Arg Gly Lys Ser Val Thr His Ser

35 40 45

ctg aga atg agt ttg agc acc gca gtc tct gat gat cat ggt gta caa 192

Leu Arg Met Ser Leu Ser Thr Ala Val Ser Asp Asp His Gly Val Gln

50 55 60

aga cgc ata gtc gag ttt cat tcc aat ctg tgg gac gac gat ttc ata 240

Arg Arg Ile Val Glu Phe His Ser Asn Leu Trp Asp Asp Asp Phe Ile

65 70 75 80

caa tct cta tca acg cct tat ggg gca cct tca tac cgt gaa cgt gct 288

Gln Ser Leu Ser Thr Pro Tyr Gly Ala Pro Ser Tyr Arg Glu Arg Ala

85 90 95

gat aga ctt att gtg gaa gta aag ggt ata ttc act tca att tca gcg 336

Asp Arg Leu Ile Val Glu Val Lys Gly Ile Phe Thr Ser Ile Ser Ala

100 105 110

gaa gat gga gaa cta atc act ccc ctc aat gat ctc att caa cgc ctt 384

Glu Asp Gly Glu Leu Ile Thr Pro Leu Asn Asp Leu Ile Gln Arg Leu

115 120 125

tta atg gtc gat aac gtt gaa cgt tta ggg att gat aga cat ttc aaa 432

Leu Met Val Asp Asn Val Glu Arg Leu Gly Ile Asp Arg His Phe Lys

130 135 140

aat gag ata aaa gca gca cta gac tat gtt tac agt tat tgg aac gaa 480

Asn Glu Ile Lys Ala Ala Leu Asp Tyr Val Tyr Ser Tyr Trp Asn Glu

145 150 155 160

aaa ggc att ggc agt gga agt gat agt ggt gtt gct gat ctc aac tca 528

Lys Gly Ile Gly Ser Gly Ser Asp Ser Gly Val Ala Asp Leu Asn Ser

165 170 175

act gcc ctg ggg ttt cga att ctt cga cta cac gga tac agt gtt tct 576

Thr Ala Leu Gly Phe Arg Ile Leu Arg Leu His Gly Tyr Ser Val Ser

180 185 190

tca gat gtg ttg gaa cac ttc aaa gag gag aag gag aag ggg cag ttt 624

Ser Asp Val Leu Glu His Phe Lys Glu Glu Lys Glu Lys Gly Gln Phe

195 200 205

gta tgt tcg gcc atc caa aca gag gaa gag ata aaa agc gtt ctg aat 672

Val Cys Ser Ala Ile Gln Thr Glu Glu Glu Ile Lys Ser Val Leu Asn

210 215 220

tta ttt cgg gcc tcc ctc att gcc ttt cct ggg gag aaa gtt atg gaa 720

Leu Phe Arg Ala Ser Leu Ile Ala Phe Pro Gly Glu Lys Val Met Glu

225 230 235 240

gag gct gaa atc ttc tct aaa ata tat tta aaa gaa gcc tta caa aat 768

Glu Ala Glu Ile Phe Ser Lys Ile Tyr Leu Lys Glu Ala Leu Gln Asn

245 250 255

att gct gtc tcc agt ctt tca cga gag ata gag tac gtt ctg gag gat 816

Ile Ala Val Ser Ser Leu Ser Arg Glu Ile Glu Tyr Val Leu Glu Asp

260 265 270

ggt tgg caa aca aat atg cca aga ttg gaa aca agg aac tac atc gat 864

Gly Trp Gln Thr Asn Met Pro Arg Leu Glu Thr Arg Asn Tyr Ile Asp

275 280 285

gta ttg gga gag aac gat cgt gat gag acg tta tat atg aac atg gag 912

Val Leu Gly Glu Asn Asp Arg Asp Glu Thr Leu Tyr Met Asn Met Glu

290 295 300

aaa ctt tta gaa att gca aaa ttg gag ttc aat att ttt cac tcc tta 960

Lys Leu Leu Glu Ile Ala Lys Leu Glu Phe Asn Ile Phe His Ser Leu

305 310 315 320

caa cag aga gag cta aaa gac ctc tcc aga tgg tgg aaa gat tcg ggt 1008

Gln Gln Arg Glu Leu Lys Asp Leu Ser Arg Trp Trp Lys Asp Ser Gly

325 330 335

ttc tct cac ctg aca ttt tct cgg cat cgt cat gtg gaa ttc tac gct 1056

Phe Ser His Leu Thr Phe Ser Arg His Arg His Val Glu Phe Tyr Ala

340 345 350

ctg gca tct tgc att gaa act gat cgc aaa cat tcc gga ttc aga ctc 1104

Leu Ala Ser Cys Ile Glu Thr Asp Arg Lys His Ser Gly Phe Arg Leu

355 360 365

ggc ttt gcc aaa atg tgt cat ctt atc acg gtt ttg gac gat ata tac 1152

Gly Phe Ala Lys Met Cys His Leu Ile Thr Val Leu Asp Asp Ile Tyr

370 375 380

gac acc ttt gga aca atg gag gag ctg gaa ctc ttc act gca gca ttt 1200

Asp Thr Phe Gly Thr Met Glu Glu Leu Glu Leu Phe Thr Ala Ala Phe

385 390 395 400

aag aga tgg gat ccg tct gcc aca gat ttg ctt cca gag tat atg aaa 1248

Lys Arg Trp Asp Pro Ser Ala Thr Asp Leu Leu Pro Glu Tyr Met Lys

405 410 415

ggg ttg tac atg gtg gtt tac gaa acc gta aat gaa att gct cga gag 1296

Gly Leu Tyr Met Val Val Tyr Glu Thr Val Asn Glu Ile Ala Arg Glu

420 425 430

gca gac aag tct caa ggc cga gag acg ctc aac gat gct cga cga gct 1344

Ala Asp Lys Ser Gln Gly Arg Glu Thr Leu Asn Asp Ala Arg Arg Ala

435 440 445

tgg gag gcc tat ctt gat tcg tat atg aaa gaa gct gag tgg atc tcc 1392

Trp Glu Ala Tyr Leu Asp Ser Tyr Met Lys Glu Ala Glu Trp Ile Ser

450 455 460

agt ggt tat ctg cca acg ttt gag gag tac atg gag acc agc aaa gtt 1440

Ser Gly Tyr Leu Pro Thr Phe Glu Glu Tyr Met Glu Thr Ser Lys Val

465 470 475 480

agt ttt ggt tat cgc ata ttc gca ttg caa ccc atc ctc act atg gat 1488

Ser Phe Gly Tyr Arg Ile Phe Ala Leu Gln Pro Ile Leu Thr Met Asp

485 490 495

gtt ccc ctt act cac cac atc ctg cag gaa ata gac ttt cca ttg agg 1536

Val Pro Leu Thr His His Ile Leu Gln Glu Ile Asp Phe Pro Leu Arg

500 505 510

ttt aat gac tta ata tgt tcc atc ctt cga ctt aaa aat gac act cgc 1584

Phe Asn Asp Leu Ile Cys Ser Ile Leu Arg Leu Lys Asn Asp Thr Arg

515 520 525

tgc tac aag gcg gac agg gcc cgt gga gaa gaa gct tcg tgt ata tcg 1632

Cys Tyr Lys Ala Asp Arg Ala Arg Gly Glu Glu Ala Ser Cys Ile Ser

530 535 540

tgt tat atg aaa gag aat cct gga tca aca gag gaa gat gct atc aat 1680

Cys Tyr Met Lys Glu Asn Pro Gly Ser Thr Glu Glu Asp Ala Ile Asn

545 550 555 560

cat atc aac gct atg gtc aat aac tta atc aaa gaa gtg aat tgg gag 1728

His Ile Asn Ala Met Val Asn Asn Leu Ile Lys Glu Val Asn Trp Glu

565 570 575

ctt ctc cga cag gac ggc acc gct cat att gct tgc aag aaa cac gct 1776

Leu Leu Arg Gln Asp Gly Thr Ala His Ile Ala Cys Lys Lys His Ala

580 585 590

ttt gac atc ctc aaa ggt tcc ctt cac ggc tac aaa tac cga gat ggg 1824

Phe Asp Ile Leu Lys Gly Ser Leu His Gly Tyr Lys Tyr Arg Asp Gly

595 600 605

ttc agc gtt gcc aac aag gaa acc aag aat tgg gtg agg aga aca gtc 1872

Phe Ser Val Ala Asn Lys Glu Thr Lys Asn Trp Val Arg Arg Thr Val

610 615 620

ctt gag tct gtg cct ttg 1890

Leu Glu Ser Val Pro Leu

625 630

<210> SEQ ID NO 78

<211> LENGTH: 630

<212> TYPE: PRT

<213> ORGANISM: Abies Grandis

<400> SEQUENCE: 78

Met Ala Leu Val Ser Ile Leu Pro Leu Ser Ser Lys Ser Val Leu His

1 5 10 15

Lys Ser Trp Ile Val Ser Thr Tyr Glu His Lys Ala Ile Ser Arg Thr

20 25 30

Ile Pro Asn Leu Gly Leu Arg Gly Arg Gly Lys Ser Val Thr His Ser

35 40 45

Leu Arg Met Ser Leu Ser Thr Ala Val Ser Asp Asp His Gly Val Gln

50 55 60

Arg Arg Ile Val Glu Phe His Ser Asn Leu Trp Asp Asp Asp Phe Ile

65 70 75 80

Gln Ser Leu Ser Thr Pro Tyr Gly Ala Pro Ser Tyr Arg Glu Arg Ala

85 90 95

Asp Arg Leu Ile Val Glu Val Lys Gly Ile Phe Thr Ser Ile Ser Ala

100 105 110

Glu Asp Gly Glu Leu Ile Thr Pro Leu Asn Asp Leu Ile Gln Arg Leu

115 120 125

Leu Met Val Asp Asn Val Glu Arg Leu Gly Ile Asp Arg His Phe Lys

130 135 140

Asn Glu Ile Lys Ala Ala Leu Asp Tyr Val Tyr Ser Tyr Trp Asn Glu

145 150 155 160

Lys Gly Ile Gly Ser Gly Ser Asp Ser Gly Val Ala Asp Leu Asn Ser

165 170 175

Thr Ala Leu Gly Phe Arg Ile Leu Arg Leu His Gly Tyr Ser Val Ser

180 185 190

Ser Asp Val Leu Glu His Phe Lys Glu Glu Lys Glu Lys Gly Gln Phe

195 200 205

Val Cys Ser Ala Ile Gln Thr Glu Glu Glu Ile Lys Ser Val Leu Asn

210 215 220

Leu Phe Arg Ala Ser Leu Ile Ala Phe Pro Gly Glu Lys Val Met Glu

225 230 235 240

Glu Ala Glu Ile Phe Ser Lys Ile Tyr Leu Lys Glu Ala Leu Gln Asn

245 250 255

Ile Ala Val Ser Ser Leu Ser Arg Glu Ile Glu Tyr Val Leu Glu Asp

260 265 270

Gly Trp Gln Thr Asn Met Pro Arg Leu Glu Thr Arg Asn Tyr Ile Asp

275 280 285

Val Leu Gly Glu Asn Asp Arg Asp Glu Thr Leu Tyr Met Asn Met Glu

290 295 300

Lys Leu Leu Glu Ile Ala Lys Leu Glu Phe Asn Ile Phe His Ser Leu

305 310 315 320

Gln Gln Arg Glu Leu Lys Asp Leu Ser Arg Trp Trp Lys Asp Ser Gly

325 330 335

Phe Ser His Leu Thr Phe Ser Arg His Arg His Val Glu Phe Tyr Ala

340 345 350

Leu Ala Ser Cys Ile Glu Thr Asp Arg Lys His Ser Gly Phe Arg Leu

355 360 365

Gly Phe Ala Lys Met Cys His Leu Ile Thr Val Leu Asp Asp Ile Tyr

370 375 380

Asp Thr Phe Gly Thr Met Glu Glu Leu Glu Leu Phe Thr Ala Ala Phe

385 390 395 400

Lys Arg Trp Asp Pro Ser Ala Thr Asp Leu Leu Pro Glu Tyr Met Lys

405 410 415

Gly Leu Tyr Met Val Val Tyr Glu Thr Val Asn Glu Ile Ala Arg Glu

420 425 430

Ala Asp Lys Ser Gln Gly Arg Glu Thr Leu Asn Asp Ala Arg Arg Ala

435 440 445

Trp Glu Ala Tyr Leu Asp Ser Tyr Met Lys Glu Ala Glu Trp Ile Ser

450 455 460

Ser Gly Tyr Leu Pro Thr Phe Glu Glu Tyr Met Glu Thr Ser Lys Val

465 470 475 480

Ser Phe Gly Tyr Arg Ile Phe Ala Leu Gln Pro Ile Leu Thr Met Asp

485 490 495

Val Pro Leu Thr His His Ile Leu Gln Glu Ile Asp Phe Pro Leu Arg

500 505 510

Phe Asn Asp Leu Ile Cys Ser Ile Leu Arg Leu Lys Asn Asp Thr Arg

515 520 525

Cys Tyr Lys Ala Asp Arg Ala Arg Gly Glu Glu Ala Ser Cys Ile Ser

530 535 540

Cys Tyr Met Lys Glu Asn Pro Gly Ser Thr Glu Glu Asp Ala Ile Asn

545 550 555 560

His Ile Asn Ala Met Val Asn Asn Leu Ile Lys Glu Val Asn Trp Glu

565 570 575

Leu Leu Arg Gln Asp Gly Thr Ala His Ile Ala Cys Lys Lys His Ala

580 585 590

Phe Asp Ile Leu Lys Gly Ser Leu His Gly Tyr Lys Tyr Arg Asp Gly

595 600 605

Phe Ser Val Ala Asn Lys Glu Thr Lys Asn Trp Val Arg Arg Thr Val

610 615 620

Leu Glu Ser Val Pro Leu

625 630

<210> SEQ ID NO 79

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 6eBamHIF

<400> SEQUENCE: 79

caattaagag atgggacccg tccgcgatgg 30

<210> SEQ ID NO 80

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 6eBamHIR

<400> SEQUENCE: 80

ccatcgcgga cgggtcccat ctcttaattg 30

<210> SEQ ID NO 81

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 9eBamHIF

<400> SEQUENCE: 81

gcatttaaga gatgggaccc gtctgccaca 30

<210> SEQ ID NO 82

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 9eBamHIR

<400> SEQUENCE: 82

ctgtggcaga cgggtcccat ctcttaaatg 30

<210> SEQ ID NO 83

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 732eNdeIF

<400> SEQUENCE: 83

cgagatgcca tacgtgaata cgcag 25

<210> SEQ ID NO 84

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 732eNde1R

<400> SEQUENCE: 84

ctgcgtattc acgtatggca tctcg 25

<210> SEQ ID NO 85

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 6-Ndel-M

<400> SEQUENCE: 85

ctgatagcaa gctcatatgg ctcttctttc 30

<210> SEQ ID NO 86

<211> LENGTH: 34

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 6-NdeI-R

<400> SEQUENCE: 86

gcccacgcgt ctcatatgag aatcagtaga tgcg 34

<210> SEQ ID NO 87

<211> LENGTH: 29

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 6-BamHI

<400> SEQUENCE: 87

cacccatagg ggatcctcag ttaatattg 29

<210> SEQ ID NO 88

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 8-NdeI-M

<400> SEQUENCE: 88

taagcgagca catatggctc tggtttcttc 30

<210> SEQ ID NO 89

<211> LENGTH: 29

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 8-BamHI

<400> SEQUENCE: 89

gcataaacgc atagcggatc ctacaccaa 29

<210> SEQ ID NO 90

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 9-NdeI-M

<400> SEQUENCE: 90

cccggggatc ggacatatgg ctcttgtttc 30

<210> SEQ ID NO 91

<211> LENGTH: 34

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 9-BamHI

<400> SEQUENCE: 91

ggtcgactct agaggatcca ctagtgatat ggat 34

<210> SEQ ID NO 92

<211> LENGTH: 27

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 11-NdeI-M

<400> SEQUENCE: 92

gaacatatgg ctctcctttc tatcgta 27

<210> SEQ ID NO 93

<211> LENGTH: 31

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 11-NdeI-R

<400> SEQUENCE: 93

ggtggtggtg tacatatgag acgcatacgg g 31

<210> SEQ ID NO 94

<211> LENGTH: 34

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 11-BamHI

<400> SEQUENCE: 94

gagactagac tggatcccat atacactgta atgg 34

<210> SEQ ID NO 95

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 2-NdeI-M

<400> SEQUENCE: 95

caaagggagc acatatggct ctgg 24

<210> SEQ ID NO 96

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 2-NdeI-R

<400> SEQUENCE: 96

ctgatgatgg tcatatgaga cgcataggtg 30

<210> SEQ ID NO 97

<211> LENGTH: 28

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 2-BamHI

<400> SEQUENCE: 97

gaccttatta ttatggatcc ggttatag 28

<210> SEQ ID NO 98

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 3NdeI-R

<400> SEQUENCE: 98

ccgatgatgg tcatatgaga cgcatgggcg 30

<210> SEQ ID NO 99

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 3-BamHI

<400> SEQUENCE: 99

gggcatagat ttgagcggat cctacaaagg 30

<210> SEQ ID NO 100

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 3e1BamHIF

<400> SEQUENCE: 100

cgtttgggaa tccatagaca tttc 24

<210> SEQ ID NO 101

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 3e1BamHIR

<400> SEQUENCE: 101

gaaatgtcta tggattccca aacg 24

<210> SEQ ID NO 102

<211> LENGTH: 26

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 3e2BamHIF

<400> SEQUENCE: 102

gaagagatgg gacccgtcct cgatag 26

<210> SEQ ID NO 103

<211> LENGTH: 26

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide PCR Primer 2-NdeI-M

<400> SEQUENCE: 103

ctatcgagga cgggtcccat ctcttc 26

<210> SEQ ID NO 104

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 3e1NdeIF

<400> SEQUENCE: 104

gaacacgaag tcctatgtga agagc 25

<210> SEQ ID NO 105

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 3e1NdeIR

<400> SEQUENCE: 105

gctcttcaca taggacttcg tgttc 25

<210> SEQ ID NO 106

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 3e3NdeIF

<400> SEQUENCE: 106

gatacgctca cttatgctcg ggaag 25

<210> SEQ ID NO 107

<211> LENGTH: 25

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide Mutagenesis Primer 3e2NdeIR

<400> SEQUENCE: 107

cttcccgagc ataagtgagc gtatc 25

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An isolated nucleic acid molecule that hybridizes under stringent conditions to a probe selected from the group consisting of the portion of SEQ ID NO: 3 extending from nucleotide 1560 to nucleotide 1694 and the portion of SEQ ID NO: 5 extending from nucleotide 1180 to nucleotide 1302.

2. An isolated nucleic acid molecule of claim 1 that hybridizes under stringent conditions to the portion of SEQ ID NO: 3 extending from nucleotide 1560 to nucleotide 1694.

3. An isolated nucleic acid molecule of claim 1 that hybridizes under stringent conditions to the portion of SEQ ID NO: 5 extending from nucleotide 1180 to nucleotide 1302.

4. An isolated nucleic acid molecule of claim 1 encoding a (−)-camphene synthase.

5. An isolated nucleic acid molecule of claim 1 encoding a gymnosperm (−)-camphene synthase.

6. An isolated nucleic acid molecule of claim 1 encoding a Grand fir (−)-camphene synthase.

7. An isolated nucleic acid molecule of claim 1 which encodes the amino acid sequence of SEQ ID NO: 65.

8. An isolated nucleic acid molecule of claim 1 having the sequence of SEQ ID NO: 64:

9. An isolated nucleic acid molecule of claim 1 encoding a (−)-β-phellandrene synthase.

10. An isolated nucleic acid molecule of claim 1 encoding a gymnosperm (−)-β-phellandrene synthase.

11. An isolated nucleic acid molecule of claim 1 encoding a Grand fir (−)-β-phellandrene synthase.

12. An isolated nucleic acid molecule of claim 1 which encodes the amino acid sequence of SEQ ID NO: 67.

13. An isolated nucleic acid molecule of claim 1 having the sequence of SEQ ID NO: 66.

14. An isolated nucleic acid molecule of claim 1 encoding a terpinolene synthase.

15. An isolated nucleic acid molecule of claim 1 encoding a gymnosperm terpinolene synthase.

16. An isolated nucleic acid molecule of claim 1 encoding a Grand fir terpinolene synthase.

17. An isolated nucleic acid molecule of claim 1 which encodes the amino acid sequence of SEQ ID NO: 78.

18. An isolated nucleic acid molecule of claim 1 having the sequence of SEQ ID NO: 77.

19. An isolated nucleic acid molecule of claim 1 encoding a (−)-limonene/(−)-α-pinene synthase.

20. An isolated nucleic acid molecule of claim 1 encoding a gymnosperm (−)-limonene/(−)-α-pinene synthase.

21. An isolated nucleic acid molecule of claim 1 encoding a Grand fir (−)-limonene/(−)-α-pinene synthase.

22. An isolated nucleic acid molecule of claim 1 which encodes the amino acid sequence of SEQ ID NO: 69.

23. An isolated nucleic acid molecule of claim 1 having the sequence of SEQ ID NO: 68.

24. An isolated nucleic acid molecule of claim 1 encoding a myrcene synthase.

25. An isolated nucleic acid molecule of claim 1 encoding a gymnosperm myrcene synthase.

26. An isolated nucleic acid molecule of claim 1 encoding a Grand fir myrcene synthase.

27. An isolated nucleic acid molecule of claim 1 which encodes the amino acid sequence of SEQ ID NO: 2.

28. An isolated nucleic acid molecule of claim 1 having the sequence of SEQ ID NO: 1.

29. An isolated nucleic acid molecule of claim 1 encoding a (−)-limonene synthase.

30. An isolated nucleic acid molecule of claim 1 encoding a gymnosperm (−)-limonene synthase.

31. An isolated nucleic acid molecule of claim 1 encoding a Grand fir (−)-limonene synthase.

32. An isolated nucleic acid molecule of claim 1 which encodes the amino acid sequence of SEQ ID NO: 6.

33. An isolated nucleic acid molecule of claim 1 having the sequence of SEQ ID NO: 5.

34. An isolated nucleic acid molecule of claim 1 encoding a (−)-pinene synthase.

35. An isolated nucleic acid molecule of claim 1 encoding a gymnosperm (−)-pinene synthase.

36. An isolated nucleic acid molecule of claim 1 encoding a Grand fir (−)-pinene synthase.

37. An isolated nucleic acid molecule of claim 1 which encodes the amino acid sequence of SEQ ID NO: 4.

38. An isolated nucleic acid molecule of claim 1 having the sequence of SEQ ID NO: 3.

39. An isolated (−)-camphene synthase protein.

40. An isolated gymnosperm (−)-camphene synthase protein of claim 39.

41. An isolated Grand fir (−)-camphene synthase protein of claim 39.

42. An isolated (−)-camphene synthase protein of claim 39, said isolated (−)-camphene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 65.

43. An isolated (−)-β-phellandrene synthase protein.

44. An isolated gymnosperm (−)-β-phellandrene synthase protein of claim 43.

45. An isolated Grand fir (−)-β-phellandrene synthase protein of claim 43.

46. An isolated (−)-β-phellandrene synthase protein of claim 43, said isolated (−)-β-phellandrene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 67.

47. An isolated terpinolene synthase protein.

48. An isolated gymnosperm terpinolene synthase protein of claim 47.

49. An isolated Grand fir terpinolene synthase protein of claim 47.

50. An isolated terpinolene synthase protein of claim 47, said isolated terpinolene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 78.

51. An isolated (−)-limonene/(−)-α-pinene synthase protein.

52. An isolated gymnosperm (−)-limonene/(−)-α-pinene synthase protein of claim 51.

53. An isolated Grand fir (−)-limonene/(−)-α-pinene synthase protein of claim 51.

54. An isolated (−)-limonene/(−)-α-pinene synthase protein of claim 51, said isolated (−)-limonene/(−)-α-pinene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 69.

55. An isolated myrcene synthase protein.

56. An isolated gymnosperm myrcene synthase protein of claim 55.

57. An isolated Grand fir myrcene synthase protein of claim 55.

58. An isolated myrcene synthase protein of claim 55, said isolated myrcene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 2.

59. An isolated (−)-limonene synthase protein.

60. An isolated gymnosperm (−)-limonene synthase protein of claim 59.

61. An isolated Grand fir (−)-limonene synthase protein of claim 59.

62. An isolated (−)-limonene synthase protein of claim 59, said isolated (−)-limonene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 6.

63. An isolated (−)-pinene synthase protein.

64. An isolated gymnosperm (−)-pinene synthase protein of claim 63.

65. An isolated Grand fir (−)-pinene synthase protein of claim 63.

66. An isolated (−)-pinene synthase protein of claim 63, said isolated (−)-pinene synthase protein comprising the amino acid sequence set forth in SEQ ID NO: 4.

67. A replicable expression vector comprising a nucleic acid sequence encoding a monoterpene synthase selected from the group consisting of (−)-camphene synthase, (−)-β-phellandrene synthase, terpinolene synthase, (−)-limonene/(−)-α-pinene synthase, myrcene synthase, (−)-limonene synthase and (−)-pinene synthase.

68. A replicable expression vector of claim 67 comprising a nucleic acid sequence encoding a (−)-camphene synthase.

69. A replicable expression vector of claim 67 comprising a nucleic acid sequence encoding a (−)-β-phellandrene synthase.

70. A replicable expression vector of claim 67 comprising a nucleic acid sequence encoding a terpinolene synthase.

71. A replicable expression vector of claim 67 comprising a nucleic acid sequence encoding a (−)-limonene/(−)-α-pinene synthase.

72. A replicable expression vector of claim 67 comprising a nucleic acid sequence encoding a myrcene synthase.

73. A replicable expression vector of claim 67 comprising a nucleic acid sequence encoding a (−)-limonene synthase.

74. A replicable expression vector of claim 67 comprising a nucleic acid sequence encoding a (−)-pinene synthase.

75. A host cell comprising a vector of claim 68.

76. A host cell comprising a vector of claim 69.

77. A host cell comprising a vector of claim 70.

78. A host cell comprising a vector of claim 71.

79. A host cell comprising a vector of claim 72.

80. A host cell comprising a vector of claim 73.

81. A host cell comprising a vector of claim 74.

82. A method of enhancing the production of a monoterpene synthase in a suitable host cell comprising introducing into the host cell an expression vector of claim 67 under conditions enabling expression of the monoterpene synthase in the host cell.

83. The method of claim 82 wherein said host cell is a plant cell.

84. The method of claim 83 wherein said cell is from a plant selected from the group consisting of Brassica, cotton, soybean, safflower, sunflower, coconut, palm, wheat, barley, rice, corn, oats, amaranth, pumpkin, squash, sesame, poppy, grape, mung beans, peanut, peas, beans, broad beans, chick peas, lentils, radish, alfalfa, cocoa, coffee, tree nuts, spinach, culinary herbs, berries, stone fruit and citrus.

85. The method of claim 83 wherein said plant cell is a seed cell.

86. The method of claim 83 wherein said plant cell is a leaf cell.

87. Seed comprising a cell produced by the method of claim 85.

88. Seed of claim 87 selected from the group consisting of seed of Brassica, cotton, soybean, safflower, sunflower, coconut, palm, wheat, barley, rice, corn, oats, amaranth, pumpkin, squash, sesame, poppy, grape, mung beans, peanut, peas, beans, broad beans, chick peas, lentils, radish, alfalfa, cocoa, coffee and tree nuts.

89. Oil extracted from the seed of claim 88.

90. Meal extracted from the seed of claim 88.