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WO1996001905A1 - Genes chimeres et procede d'augmentation de la teneur en threonine de graines de plantes - Google Patents

Genes chimeres et procede d'augmentation de la teneur en threonine de graines de plantes Download PDF

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Publication number
WO1996001905A1
WO1996001905A1 PCT/US1995/008501 US9508501W WO9601905A1 WO 1996001905 A1 WO1996001905 A1 WO 1996001905A1 US 9508501 W US9508501 W US 9508501W WO 9601905 A1 WO9601905 A1 WO 9601905A1
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Prior art keywords
seeds
gene
plant
plants
sequence
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PCT/US1995/008501
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English (en)
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Saverio Carl Falco
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E.I. Du Pont De Nemours And Company
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Priority to BR9510174A priority Critical patent/BR9510174A/pt
Priority to MX9606384A priority patent/MX9606384A/es
Priority to AU29636/95A priority patent/AU2963695A/en
Priority to EP95925537A priority patent/EP0769061A1/fr
Publication of WO1996001905A1 publication Critical patent/WO1996001905A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)

Definitions

  • This invention relates to a chimeric gene encoding a bifunctional feedback- insensitive aspartokinase-homoserine dehydrogenase (AK-HDH), which is operably linked to a plant chloroplast transit sequence, and to plant seed-specific regulatory sequences.
  • AK-HDH bifunctional feedback- insensitive aspartokinase-homoserine dehydrogenase
  • lysine Human food and animal feed, derived from many grains, are deficient in essential amino acids, such as lysine, the sulfur amino acids methionine and cysteine, threonine and tryptophan.
  • threonine is the third most limiting amino acid, after lysine and the sulfur amino acids, for the dietary requirements of many animals.
  • Threonine along with methionine, lysine and isoleucine, are amino acids derived from aspartate.
  • the first step in the biosynthetic pathway is the phosphorylation of aspartate by the enzyme aspartokinase (AK), and this enzyme has been found to be an important target for regulation of the pathway in many organisms.
  • AK aspartokinase
  • the aspartate family pathway is also believed to be regulated at the branch-point reactions.
  • HDH homoserine dehydrogenase
  • Galili and co-workers have reported on the introduction of an E. coli lysC gene that encodes a lysine-sensitive AK enzyme into tobacco cells via transformation [Galili et al. (1992) Eur. Patent Appl. 91119328.2; Shaul et al. (1992) Plant Physiol. i 00:1157-1163]. Expression of the E. coli enzyme resulted in small increases in the levels of free threonine in d e leaves and seeds of transformed plants, but effects on the total tlireonine content were too small to be detected. Falco isolated a mutant of the E. coli lysC gene, which encoded a lysine- insensitive AK.
  • This inventions concerns a chimeric gene wherein a nucleic acid fragment encoding a bi-fimctional protein with aspartokinase and homoserine dehydrogenase activities, both of which are substantially insensitive to end-product inhibition, is operably linked to a plant chloroplast transit sequence and to a seed-specific regulatory sequence.
  • the nucleic acid fragment comprises the E. coli metL gene.
  • This invention also concerns a plant comprising in its genome the chimeric gene described above and seeds obtained from that plant. This invention further relates to a method for increasing the threonine content of the seeds of plants comprising:
  • step (b) growing fertile mature plants from the transformed plant cells obtained from step (a) under conditions suitable to obtain seeds;
  • step (c) selecting from the progeny seed of step (b) for those seeds containing increased levels of threonine compared to untransformed seeds. Also disclosed are seeds obtained by this method and plants obtained from such seeds.
  • Figure 1 shows a map of plasmid pBT718.
  • Figure 2 shows a map of plasmid pBT726.
  • Figure 3 shows a map of plasmid pBT727.
  • Figure 4 shows a map of plasmid pBT728.
  • Figure 5 shows a map of plasmid pBT733.
  • Figure 6 shows a map of plasmid pBT766.
  • SEQ ID NOS: 1-2 were used in Example 1 as PCR primers to isolate and modify the E. coli metL gene.
  • SEQ ID NOS:3-8 were used in Example 2 to create a corn chloroplast transit sequence and link the sequence to the E. coli metL gene.
  • SEQ ID NO:9 was used in Example 4 to create a soybean chloroplast transit sequence and link the sequence to the E. coli metL gene.
  • nucleic acid fragments refers to a large molecule which can be single- stranded or double-stranded, composed of monomers (nucleotides) containing a sugar, phosphate and either a purine or pyrimidine.
  • a "nucleic acid fragment” is a fraction of a given nucleic acid molecule.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • a “genome” is the entire body of genetic material contained in each cell of an organism.
  • nucleotide sequence refers to a polymer of DNA or RNA which can be single- or double- stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • DNA sequences that may involve base changes that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. It is therefore understood that the invention encompasses more than the specific exemplary sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changes that do not substantially affect the functional properties of the resulting protein molecule are also contemplated.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine can also be expected to produce a biologically equivalent product.
  • Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on the biological activity of the protein.
  • Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.
  • "essentially similar" sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65°C), with the sequences exemplified herein.
  • Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding) and following (3 1 non- coding) the coding region.
  • “Native” gene refers to the gene as found in nature with its own regulatory sequences.
  • “Chimeric” gene refers to a gene comprising heterogeneous regulatory and coding sequences.
  • “Endogenous” gene refers to the native gene normally found in its natural location in the genome.
  • a “foreign” gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.
  • Coding sequence refers to a DNA sequence that codes for a specific protein and excludes the non-coding sequences.
  • “Initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation). "Open reading frame” refers to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
  • RNA transcript refers to the product resulting from RNA polymerase- catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript.
  • Messenger RNA (mRNA) refers to RNA that can be translated into protein by the cell.
  • cDNA refers to a double-stranded DNA, one strand of which is complementary to and derived from mRNA by reverse transcription.
  • Sense RNA refers to RNA transcript that includes the mRNA.
  • regulatory sequences refer to nucleotide sequences located upstream (5'), within, and/or downstream (3') to a coding sequence, which control the transcription and/or expression of the coding sequences, potentially in conjunction with the protein biosynthetic apparatus of the cell.
  • regulatory sequences include promoters, translation leader sequences, transcription termination sequences, and polyadenylation sequences.
  • Promoter refers to a DNA sequence in a gene, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
  • a promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. It may also contain enhancer elements.
  • an “enhancer” is a DNA sequence which can stimulate promoter activity. It may be an innate element of the promoter or a heterologous element inserted to enhance the level and/or tissue-specificity of a promoter. "Constitutive promoters” refers to those that direct gene expression in all tissues and at all times. "Organ- specific” or “development-specific” promoters as referred to herein are those that direct gene expression almost exclusively in specific organs, such as leaves or seeds, or at specific development stages in an organ, such as in early or late embryogenesis, respectively.
  • operably linked refers to nucleic acid sequences on a single nucleic acid molecule which are associated so that the function of one is affected by the other.
  • a promoter is operably linked with a structure gene (i.e., a gene encoding aspartokinase that is lysine-insensitive as given herein) when it is capable of affecting the expression of that structural gene (i.e., that the structural gene is under the transcriptional control of the promoter).
  • expression is intended to mean the production of the protein product encoded by a gene. More particularly, “expression” refers to the transcription and stable accumulation of the sense (mRNA) or tha antisense RNA derived from the nucleic acid fragment(s) of the invention that, in conjuction with the protein apparatus of the cell, results in altered levels of protein product. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of preventing the expression of the target protein. "Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.
  • altered levels refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
  • the "3' non-coding sequences” refers to the DNA sequence portion of a gene that contains a polyadenylation signal and any other regulatory signal capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • translation leader sequence refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon.
  • the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
  • “Mature” protein refers to a post-translationally processed polypeptide without its targeting signal.
  • Precursor protein refers to the primary product of translation of mRNA.
  • a “chloroplast targeting signal” is an amino acid sequence which is translated in conjunction with a protein and directs it to the chloroplast.
  • Chloroplast transit sequence refers to a nucleotide sequence that encodes a chloroplast targeting signal.
  • End-product inhibition or “feedback inhibition” refers to a biological regulatory mechanism wherein the catalytic activity of an enzyme in a biosynthetic pathway is reversibly inhibited by binding to one or more of the end-products of the pathway when the concentration of the end-product(s) reaches a sufficiently high level, thus slowing the biosynthetic process and preventing over-accumulation of the end-product.
  • Transformation herein refers to the transfer of a foreign gene into the genome of a host organism and its genetically stable inheritance.
  • methods of plant transformation include Agrobacterium -mediated transfo ⁇ nation and particle-accelerated or "gene gun” transformation technology.
  • “Host cell” means the cell that is transformed with the introduced genetic material.
  • AK-HDH Genes This invention concerns chimeric genes encoding bi-functional AK-HDH enzymes, wherein both catalytic activities are insensitive to end-product inhibition.
  • Over-expression of feedback-insensitive AK increases flux through the entire pathway of aspartate-derived amino acids even in the presence of high concentrations of the pathway end-products lysine, threonine and methionine.
  • Over-expression of a bifunctional feedback-insensitive AK-HDH enzyme directs the increased flux through the threonine branch of the aspartate-derived amino acid pathway, increasing the rate of threonine biosynthesis.
  • a number of AK and AK-HDH genes have been isolated and sequenced. These include the thrA gene of E.
  • E. coli (Katinka et al. (1980) Proc. Natl. Acad. Sci. USA 77:5730-5733], the metL gene of E. coli (Zakin et al. (1983) /. Biol. Chem. 23 ⁇ °:3028-3031], the lysC gene of E. c ⁇ 7[Cassan et al. (1986) /. Biol. Chem. 2(5 :1052-1057], and the HOM3 gene of 5. cerevisiae [Rafalski et al. (1988) J. Biol. Chem. 2(55:2146-2151].
  • the thrA gene of E. coli encodes a bifunctional protein, AKI-HDHI.
  • the AK and HDH activities of this protein is inhibited by threonine.
  • the metL gene of E. coli also encodes a bifunctional protein, AKII-HDHII, and both the AK and HDH activities of this protein are insensitive to all pathway end-products.
  • the E. coli lysC gene encodes AKIH, which is sensitive to lysine inhibition.
  • the HOM3 gene of yeast encodes an AK which is sensitive to threonine.
  • E. coli metL gene encoding AKII-HDHII is preferred. As indicated above, this gene has been isolated and sequenced. Thus, it can be easily obtained from E. coli genomic DNA by a variety of techniques well known to those skilled in the art, for example via PCR using oligonucleotide primers based on the published DNA sequence.
  • Plant mutants that express lysine-insensitive AK-HDH are known.
  • lysine plus threonine-resistant mutants bearing mutations in two unlinked genes that result in two different lysine-insensitive AK isoenzymes have been described [Bright et al. (1982) Nature 299:278-279, Rognes et al. (1983) Planta 257:32-38, Arruda et al. (1984) Plant Phsiol. 7(5:442-446].
  • a lysine plus threonine- resistant cell line had AK activity that was less sensitive to lysine inhibition than its parent line [Hibberd et al.
  • AK-HDH in the Seeds of Plants In order to increase biosynthesis of threonine in seeds, suitable regulatory sequences are provided to create chimeric genes for high level seed-specific expression of the AK-HDH coding region.
  • the replacement of the native regulatory sequences accomplishes two things: 1) any pleiotropic effects that the accumulation of excess free threonine might have on the vegetative growth of plants is prevented because the chimeric gene(s) is not expressed in vegetative tissue of the transformed plants 2) high level expression of the enzyme(s) is obtained in the seeds.
  • the expression of foreign genes in plants is well-established [De Blaere et al.
  • AK-HDH mRNA Proper level of expression of AK-HDH mRNA may require the use of different chimeric genes utilizing different promoters. Such chimeric genes can be transferred into host plants either together in a single expression vector or sequentially using more than one vector.
  • a preferred class of heterologous hosts for the expression of AK-HDH genes are eukaryotic hosts, particularly the cells of higher plants. Particularly preferred among the higher plants and the seeds derived from them are soybean, rapeseed (Brassica napus, B.
  • campestris sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn, tobacco (Nicotiana Tubacum), alfalfa (Medicago sativa), wheat (Triticum sp), barley (Hordeum vulgare), oats (Avena sativa, L), sorghum (Sorghum bicolor), rice (Oryza sativa), and forage grasses. Expression in plants will use regulatory sequences functional in such plants.
  • the origin of the promoter chosen to drive the expression of the coding sequence is not critical as long as it has sufficient transcriptional activity to accomplish the invention by expressing translatable mRNA for AK-HDH genes in the desired host tissue.
  • Preferred promoters are those that allow expression of the protein specifically in seeds. This may be especially useful, since seeds are the primary source of vegetable amino acids and also since seed-specific expression will avoid any potential deleterious effect in non-seed organs.
  • seed-specific promoters include, but are not limited to, the promoters of seed storage proteins.
  • the seed storage proteins are strictly regulated, being expressed almost exclusively in seeds in a highly organ-specific and stage-specific manner [Higgins et al.(1984) Ann. Rev. Plant Physiol. 55:191-221; Goldberg et al.(1989) Cell 5(5:149-160; Thompson et al. (1989) BioEssays 20:108-113].
  • different seed storage proteins may be expressed at different stages of seed development.
  • seed-specific expression of seed storage protein genes in transgenic dicotyledonous plants include genes from dicotyledonous plants for bean ⁇ -phaseolin [Sengupta-Goplalan et al. (1985) Proc. Natl. Acad. Sci. USA 52:3320-3324; Hoffman et al. (1988) Plant Mol. Biol. 27:717-729], bean lectin [Voelker et al. (1987) EMBO J. 6: 3571-3577], soybean lectin [Okamuro et al. (1986) Proc. Natl. Acad. Sci.
  • soybean kunitz trypsin inhibitor [Perez-Grau et al. (1989) Plant Cell 1 :095-l 109], soybean ⁇ -conglycrnin [Beachy et al. (1985) EMBO J. 4:3047-3053; Barker et al. (1988) Proc. Natl. Acad. Sci. USA 55:458-462; Chen et al. (1988) EMBO J. 7:297-302; Chen et al. (1989) Dev. Genet. 20:112-122; Naito et al. (1988) Plant Mol. Biol. 22:109-123], pea vicilin [Higgins et al. (1988) Plant Mol. Biol. 22:683-695], pea convicilin [Newbigin et al. (1990) Planta 250:461], pea legumin [Shirsat et al.
  • promoters of seed-specific genes also maintain their temporal and spatial expression pattern in transgenic plants.
  • Such examples include linking either the Phaseolin or Arabidopsis 2S albumin promoters to the Brazil nut 2S albumin coding sequence and expressing such combinations in tobacco, Arabidopsis, or Brassica napus [Altenbach et al., (1989) Plant Mol. Biol. 25:513-522; Altenbach et al., (1992) Plant Mol. Biol. 25:235-245; De Clercq et al.,
  • nucleic acid fragment of the invention will be the heterologous promoters from several extensively- characterized soybean seed storage protein genes such as those for the Kunitz trypsin inhibitor [Jofuku et al. (1989) Plant Cell 2:1079-1093; Perez-Grau et al. (1989) Plant Cell 2:1095-1109], glytinin [Nielson et al. (1989) Plant Cell 2:313-328], ⁇ -conglycinin [Harada et al. (1989) Plant Cell 2:415-425].
  • Promoters of genes for ⁇ '- and ⁇ -subunits of soybean ⁇ -conglycinin storage protein will be particularly useful in expressing AK-HDH mRNA in the cotyledons at mid- to late- stages of soybean seed development [Beachy et al. (1985) EMBO J. 4:3047-3053; Barker et al. (1988) Proc. Natl. Acad. Sci. USA 55:458 ⁇ 62; Chen et al. (1988) EMBO J. 7:297-302; Chen et al. (1989) Dev. Genet. 20:112-122; Naito et al.
  • the two promoters show different temporal regulation: the promoter for the ⁇ '-subunit gene is expressed a few days before that for the ⁇ -subunit gene.
  • the promoters from several extensively characterized corn seed storage protein genes such as endosperm-specific promoters from the 10 kD zein [Kirihara et al. (1988) Gene 72:359-370], the 27 kD zein [Prat et al.
  • enhancers or enhancer-like elements into other promoter constructs will also provide increased levels of primary transcription for AK-HDH genes to accomplish the invention.
  • enhancers or enhancer-like elements include viral enhancers such as that found in the 35S promoter [Odell et al. (1988) Plant Mol. Biol. 20:263-272], enhancers from the opine genes [Fromm et al.
  • any 3' non-coding region capable of providing a polyadenylation signal and other regulatory sequences that may be required for the proper expression of the AK-HDH coding regions can be used to accomplish the invention.
  • any storage protein such as the 3' end of the bean phaseolin gene, the 3' end of the soybean ⁇ -conglycinin gene, the 3' end from viral genes such as the 3' end of the 35S or the 19S cauliflower mosaic virus transcripts, the 3' end from the opine synthesis genes, the 3' ends of ribulose 1,5-bisphosphate carboxylase or chloro
  • DNA sequences coding for intracellular localization sequences may be added to the AK-HDH coding sequence if required for the proper expression of the proteins to accomplish the invention.
  • Plant amino acid biosynthetic enzymes are known to be localized in the chloroplasts and therefore are synthesized with a chloroplast targeting signal.
  • Bacterial proteins such as E. coli AKH-HDHII have no such signal.
  • a chloroplast transit sequence could, therefore, be fused to the coding sequence.
  • Preferred chloroplast transit sequences are those of the small subunit of ribulose 1,5-bisphosphate carboxylase, e.g. from soybean [Berry-Lowe et al. (1982) /. Mol. Appl. Genet.
  • eukaryotic cells i.e., of transformation
  • Such methods include those based on transformation vectors utilizing the Ti and Ri plasmids of Agrobacterium spp. It is particularly preferred to use the binary type of these vectors.
  • Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton and rape [Pacciotti et al. (1985) Bio/Technology 5:241; Byrne et al.
  • a seed meal can be prepared by any of a number of suitable methods known to those skilled in the art.
  • the seed meal can be partially or completely defatted, via hexane extraction for example, if desired.
  • Protein extracts can be prepared from the meal and analyzed for AK or HDH enzyme activities. Altematively the presence of any of the proteins can be tested for immunologically by methods well-known to those skilled in the art.
  • free amino acids can be extracted from the meal and analyzed by methods known to those skilled in the art [Bieleski et al. (1966) Anal.
  • Amino acid composition can then be determined using any commercially available amino acid analyzer.
  • meal containing both protein-bound and free amino acids can be acid hydrolyzed to release the protein-bound amino acids and the composition can then be determined using any commercially available amino acid analyzer. Seeds expressing the AK-HDH protein and with higher threonine content than the wild type seeds can thus be identified and propagated.
  • EXAMPLE 1 Isolation of the E. coli metL Gene and Over-Expression if AKII-HDHII in E. coli
  • the metL gene of E. coli encodes a bifunctional protein, AKII-HDHII; the AK and HDH activities of this enzyme are insensitive to all pathway end-products.
  • the metL gene of E. coli has been isolated and sequenced previously [Zakin et al. (1983) /. Biol. Chem. 255:3028-3031].
  • a DNA fragment containing the metL gene was isolated and modified from E. coli genomic DNA obtained from strain LE392 using PCR.
  • the following PCR primers were designed and synthesized:
  • CF23 SEQ ID NO:l: 5'-GAAACCATGG CCAGTGTG AT TGCGCAGGCA-3'
  • CF24 SEQ ID NO:2:
  • primers add an Nco I site which includes a translation initiation codon at the amino terminus of the AKII-HDHII protein.
  • restriction site and additional codon GCC coding for alanine, was also added to the amino terminus of the protein.
  • the primers also add a Kpn I site immediately following the translation stop codon.
  • PCR was performed using a Perkin-Elmer Cetus kit according to the instructions of the vendor on a thermocycler manufactured by the same company. The primers were at a concentration of 10 ⁇ M and the thermocy cling conditions were:
  • an expression vector base uponpET-3a [Rosenberg et al. (1987) Gene 56:125-135] which employs the bacteriophage T7 RNA polymerase/T7 promoter system was constructed. First the EcoR I and Hind in sites in pET-3a were destroyed at their original positions by cutting, filling the ends using the Klenow fragment of DNA polymerase and religating. An oligonucleotide adaptor containing EcoR I and Hind IH sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector.
  • Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis.
  • the DNA sequence of pET-3aM in this region, 5'-CATATGG. was converted to 5'-CCCATGG. creating plasmid pBT430.
  • This plasmid was further modified by additon of a Kpn I site downstream of the Nco I site using oligonucleotide adaptors.
  • the 2.4 kb Nco I and Kpn I metL fragments described above were inserted into the modified pBT430 expression vector cut with Nco I and Kpn I.
  • DNA was isolated from 8 clones carrying the 2.4 kb fragment in the expression vector and transformed into the expression host strain BL21(DE3). Cultures were grown in TB medium containing ampicillin (100 mg/L) at
  • the cells were collected by centrifugation and resuspended in l/25th the original culture volume in 50 mM NaCl; 50 mM Tris-Cl, pH 7.5; 1 mM EDTA, and frozen at -20°C, thawed at 37°C and sonicated, in an ice-water bath, to lyse the cells.
  • the lysate was centrifuged at 4°C for 5 min at 12,000 rpm. The supernatant was removed and the pellet was resuspended in the above buffer.
  • the supernatant fractions were assayed for HDH enzyme activities to identify clones expressing functional proteins. HDH activity was assayed as shown below:
  • AK activity was assayed as shown below:
  • AK ASSAY Assay mix (for 12 X l.OmL or 48 X 0.25mL assays): 2.5 mL H2O 2.0 mL4M KOH 2.0 mL4M NH 2 OH-HCl 1.0 mL 1M Tris-HCl pH 8.0 0.5 mL 0.2M ATP (121 mg/mL in 0.2M NaOH)
  • Each 1.5 mL eppendorf assay tube contains:
  • FeC reagent is: 10% w/v FeCl3 50 g
  • the major protein visible by Coomassie blue staining in both the pellet and supernatant fractions had a molecular weight of about 85 kd, the expected size for AKII-HDHII.
  • the metL gene in plasmid pBT718 ( Figure 1) from clone 5 was used for all subsequent work.
  • AKII-HDHII protein derived from clone 5 was sent to Hazelton Research Facility (310 Swampridge Road, Denver, PA 17517) to have rabbit antibodies raised against the protein.
  • the globulin 1 promoter and 3' sequences were isolated from a Clontech com genomic DNA library using oligonucleotide probes based on the published sequence of the globulin 1 gene [Kriz et al. (1989) Plant Physiol. 92:636].
  • the cloned segment includes the promoter fragment extending 1078 nucleotides upstream from the ATG translation start codon, the entire globulin coding sequence including introns and the 3' sequence extending 803 bases from the translational stop.
  • Nco I site was introduced at the ATG start codon, and Kpn I and Xba I sites were introduced following the translational stop codon via PCR to create vector pCC50. There is a second Nco I site within the globulin 1 promoter fragment.
  • the globulin 1 gene cassette is flanked by Hind HI sites.
  • the glutelin 2 promoter was cloned from com genomic DNA using PCR with primers based on the published sequence [Reina et al. (1990) Nucleic Acids Res. 75:6426-6426].
  • the promoter fragment includes 1020 nucleotides upstream from the ATG translation start codon.
  • An Nco I site was introduced via PCR at the ATG start site to allow for direct translational fusions.
  • a BamH I site was introduced on the 5' end of the promoter.
  • the 1.02 kb BamH I to Nco I promoter fragment was cloned into the BamH I to Nco I sites of a previously constructed plant expression vector replacing the 35S promoter to create vector pML90. This vector contains the glutelin 2 promoter linked to the GUS coding region and the NOS 3'.
  • Plant amino acid biosynthetic enzymes are known to be localized in the chloroplasts and therefore are synthesized with a chloroplast targeting signal. Bacterial proteins have no such signal.
  • a chloroplast transit sequence (cts) was therefore fused to the E. coli metL coding sequence in the chimeric genes described below.
  • the cts used was based on the the cts of the small subunit of ribulose 1,5-bisphosphate carboxylase from com [Lebrun et al. (1987) Nucleic Acids Res. 75:4360] and is designated mcts.
  • Oligonucleotides SEQ ID NO:3 and SEQ ID NO:4, which encode the carboxy terminal part of the com chloroplast targeting signal, were annealed, resulting in Xba I and Nco I compatible ends, purified via polyacrylamide gel electrophoresis, and inserted into Xba I plus Nco I digested pBT718 ( Figure 1). The insertion of the correct sequence was verified by DNA sequencing yielding pBT725. To complete the co chloroplast targeting signal, the amino terminal part of the com chloroplast targeting signal from a previous construct, pBT580, was inserted.
  • the plasmid pBT580 was constructed as follows: oligonucleotides SEQ ID NO:3 and SEQ ID NO:4 (above) were annealed, resulting in Xba I and Nco I compatible ends, purified via polyacrylamide gel electrophoresis, and inserted into an Xba I plus Nco I digested plasmid containing the E. coli lysC gene, thus fusing the carboxy terminal part of the com chloroplast targeting signal to the AKIH protein, and destroying the Nco I site. The insertion of the correct sequence was verified by DNA sequencing yielding pBT556.
  • Oligonucleotides SEQ ID NO:5 and SEQ ID NO:6, which encode the middle part of the chloroplast targeting signal, were annealed, resulting in Bgl II and Xba I compatible ends, purified via polyacrylamide gel electrophoresis, and inserted into Bgl II and Xba I digested pBT556. The insertion of the correct sequence was verified by DNA sequencing yielding pBT557.
  • Oligonucleotides SEQ ID NO:7 and SEQ ID NO:8, which encode the amino terminal part of the chloroplast targeting signal, were annealed, resulting in Nco I and Afl II compatible ends, purified via polyacrylamide gel electrophoresis, and inserted into Nco I and Afl II digested pBT557. The insertion of the correct sequence was verified by DNA sequencing yielding pBT558. Thus the complete mcts was fused to the lysC gene.
  • the mcts ⁇ ysC-M4 coding sequence was isolated from plasmid pBT558 and inserted into Nco I plus Sma I digested pML90 (above) creating plasmid pBT580.
  • pBT580 was digested with BamH I and Xba I yielding a 1.14 kb fragment containing the glutelin 2 promoter plus the amino terminal part of the corn chloroplast targeting signal. This fragment was inserted into pBT725 digested with Bgl II and Xba I, creating pBT726 ( Figure 2) wherein the complete mcts was fused to the metL coding sequence.
  • globulin 1 promoter/mcts/metL/globulin 1 3' region the 2.6 kb Nco I to Kpn I fragment containing the mcts/metL coding sequence was isolated from plasmid pBT726 and inserted into Nco I (partial digest) plus Kpn I digested pCC50 creating plasmid ⁇ BT727 ( Figure 3).
  • the bar gene was driven by the 35S promoter from Cauliflower Mosaic Virus and uses the termination and polyadenylation signal from the octopine synthase gene from Agrobacterium tumefaciens.
  • Embryogenic callus cultures were initiated from immature embryos (about 1.0 to 1.5 mm) dissected from kernels of a com line bred for giving a "type II callus" tissue culture response.
  • the embryos were dissected 10 to 12 d after pollination and were placed with the axis-side down and in contact with agarose- solidified N6 medium [Chu et al. (1974) Sci Sin 25:659-668] supplemented with 1.0 mg/L 2,4-D (N6-1.0). The embryos were kept in the dark at 27°C.
  • Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryos and somatic embryos borne on suspensor structures proliferated from the scutellum of the immature embryos.
  • Clonal embryogenic calli isolated from individual embryos were identified and sub-cultured on N6-1.0 medium every 2 to 3 weeks.
  • the particle bombardment method was used to transfer genes to the callus culture cells.
  • a BiolisticTM PDS-1000/He BioRAD Laboratories, Hercules, CA was used for these experiments.
  • Circular plasmid DNA or DNA which had been linearized by restriction endonuclease digestion was precipitated onto the surface of gold particles.
  • DNA from two or three different plasmids, one containing the selectable marker for com transformation, and one contaixiing the chimeric gene for increased threonine accumulation in seeds were co-precipitated.
  • 2.5 ⁇ g of each DNA in water at a concentration of about 1 mg/mL
  • 25 ⁇ L of gold particles average diameter of 1.0 ⁇ m
  • Embryogenic callus was arranged in a circular area of about 4 cm in diameter in the center of a 100 X 20 mm petri dish containing N6-1.0 medium supplemented with 0.25M sorbitol and 0.25M mannitol.
  • the tissue was placed on this medium for 4-6 h prior to bombardment as a pretreatment and remained on the medium during the bombardment procedure.
  • the petri dish containing the tissue was placed in the chamber of the PDS-1000/He. The air in the chamber was then evacuated to a vacuum of 28-29 inch of Hg.
  • the macrocarrier was accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1080-1100 psi.
  • the tissue was placed approximately 8 cm from the stopping screen. Five to seven plates of tissue were bombarded with the DNA-coated gold particles. Following bombardment, the callus tissue was transferred to N6-1.0 medium without supplemental sorbitol or mannitol.
  • tissue was transferred to selective medium, N6-1.0 medium that contained 2 mg/L bialaphos. All tissue was transferred to fresh N6-1.0 medium supplemented with bialaphos every 2 weeks. After 6-12 weeks clones of actively growing callus were identified. Callus was then transferred to an MS-based medium that promotes plant regeneration.
  • phaseolin 5' region/cts/metL/phaseolin 3' region A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the ⁇ subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris [Doyle et al. (1986) /. Biol. Chem. 261:9228-9238] was used for expression in transformed soybean.
  • the phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin.
  • Nco I which includes the ATG translation initiation codon
  • Sma I which includes the ATG translation initiation codon
  • Kpn I The entire cassette is flanked by Hind HI sites.
  • Plant amino acid biosynthetic enzymes are known to be localized in the chloroplasts and therefore are synthesized with a chloroplast targeting signal.
  • the bacterial protein AKLI-HDHII has no such signal.
  • a chloroplast transit sequence (cts) was therefore fused to the E. coli metL coding sequence in the chimeric gene.
  • the cts used, SEQ ID NO:9: was equivalent to the the cts of the small subunit of ribulose 1,5-bisphosphate carboxylase from soybean [Berry-Lowe et al. (1982) J. Mol. Appl. Genet. 2:483-498].
  • the cts was flanked with Nco I and inserted into the metL seed expression cassette of pBT733 creating plasmid pBT766 ( Figure 6).
  • Soybean embryogenic suspension cultures were maintained in 35 mL liquid media (SB55) on a rotary shaker, 150 rpm, at 26°C with florescent lights on a 16:8 hour day/night schedule. Cultures were subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.
  • Soybean embryogenic suspension cultures were transformed by the method of particle gun bombardment [Kline et al. (1987) Nature (London) 527:70, U.S. Patent No. 4,945,050].
  • a Du Pont BiolisticTM PDS1000 HE instrument (helium retrofit) was used for these transformations.
  • the selectable marker gene for soybean transformation was a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Virus [Odell et al.(1985) Nature 525:810-812], the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli) [Gritz et al.(1983) Gene 25:179-188] and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.
  • the seed expression cassette, phaseolin 5' region/cts/metL/phaseolin 3' region, (Example 4) was isolated as an approximately 4.5 kb Hind HI fragment from pBT766 ( Figure 6). This fragment was inserted into a unique Hind WL site of the vector carrying the marker gene creating plasmid pBT767.
  • To 50 ⁇ L of a 60 mg/mL 1 ⁇ gold particle suspension was added (in order); 5 ⁇ L DNA (1 ⁇ g/ ⁇ L), 20 ⁇ l spermidine (0.1 M), and 50 ⁇ L CaCl2 (2.5 M). The particle preparation was agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed.
  • the DNA-coated particles were then washed once in 400 ⁇ L 70% ethanol and resuspended in 40 ⁇ L of anhydrous ethanol.
  • the DNA/particle suspension was sonicated three times for one second each. Five ⁇ L of the DNA-coated gold particles were then loaded on each macro carrier disk.
  • B5 Vitimin Stock SB 55 10 g m-inositol 10 mL each MS stocks 100 mg nicotinic acid 1 mL B5 Vitamin stock 100 mg pyridoxine HC1 0.8 g NH NO3 1 g thiamine 3.033 g KNO 3
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

Gène chimère codant une aspartokinase-homosérine déshydrogénase (AK-HDH) bifonctionnelle insensible aux rétroactions, qui est lié de manière active à une séquence de transit de chloroplaste de plante et à des séquences régulatrices spécifiques des graines de plantes. Ledit gène chimère est transformé dans des plantes, des quantités accrues de thréonine libre s'accumulant dans les graines.
PCT/US1995/008501 1994-07-08 1995-07-06 Genes chimeres et procede d'augmentation de la teneur en threonine de graines de plantes WO1996001905A1 (fr)

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BR9510174A BR9510174A (pt) 1994-07-08 1995-07-06 Gene quimérico planta sementes método para aumentar o teor de treonina das sementes de plantas
MX9606384A MX9606384A (es) 1994-07-08 1995-07-06 Genes quimericos y metodo para incrementar el contenido de treonina de las semillas de plantas.
AU29636/95A AU2963695A (en) 1994-07-08 1995-07-06 Chimeric genes and method for increasing the threonine content of the seeds of plants
EP95925537A EP0769061A1 (fr) 1994-07-08 1995-07-06 Genes chimeres et procede d'augmentation de la teneur en threonine de graines de plantes

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WO1998050569A3 (fr) * 1997-05-05 1999-02-18 Dow Agrosciences Llc Sequences nucleotidiques des genes codant pour la thio-esterase de la proteine transporteuse d'acyles d'oleoyle du mais et pour la thio-esterase de la proteine transporteuse d'acyles de palmitoyle et leur utilisation pour modifier la teneur en acides gras d'une huile
US6331664B1 (en) 1997-05-05 2001-12-18 Dow Agrosciences Llc Acyl-ACP thioesterase nucleic acids from maize and methods of altering palmitic acid levels in transgenic plants therewith
WO1998055601A3 (fr) * 1997-06-06 1999-03-04 Du Pont Enzymes de biosynthese d'acides amines d'origine vegetale
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WO1998056935A3 (fr) * 1997-06-12 1999-03-11 Du Pont Enzymes biosynthetiques d'acides amines vegetaux
WO1999040209A1 (fr) * 1998-02-09 1999-08-12 Pioneer Hi-Bred International, Inc. Modification de compositions d'acides amines dans des graines
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AU2963695A (en) 1996-02-09
ZA955629B (en) 1997-01-06
CA2192550A1 (fr) 1996-01-25
EP0769061A1 (fr) 1997-04-23
HUT77112A (hu) 1998-03-02
BR9510174A (pt) 1997-11-04
MX9606384A (es) 1997-03-29

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