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WO1991004325A1 - Proteine associee a la membrane de plantes, qui possede une activite de reductase du nitrate - Google Patents

Proteine associee a la membrane de plantes, qui possede une activite de reductase du nitrate Download PDF

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Publication number
WO1991004325A1
WO1991004325A1 PCT/US1990/004397 US9004397W WO9104325A1 WO 1991004325 A1 WO1991004325 A1 WO 1991004325A1 US 9004397 W US9004397 W US 9004397W WO 9104325 A1 WO9104325 A1 WO 9104325A1
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activity
nitrate reductase
plant
fractions
membrane
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PCT/US1990/004397
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English (en)
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Michael R. Ward
Rudolf Tischner
Ray C. Huffaker
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The General Hospital Corporation
The Regents Of The University Of California
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Publication of WO1991004325A1 publication Critical patent/WO1991004325A1/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.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • 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.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
    • C12Y107/01—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with NAD+ or NADP+ as acceptor (1.7.1)
    • C12Y107/01001—Nitrate reductase (NADH) (1.7.1.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • a membrane associated protein from plants which has nitrate reductase activity is provided.
  • the invention pertains to a nitrate reductase activity of plants, and in particular of barley seedlings.
  • the invention further pertains to recombinant molecules which express this activity, to transformed plants which express such activity, and to the uses of this activity in agriculture. This invention was made in part with support from grant
  • Nitrate (NO3 " ) is the major source of nitrogen for higher plants. Such nitrate must be transported into the plant's cells, and then chemically reduced to ammonia, in order to be incorporated into amino acids. The transport of NO3 " by roots is, thus, the first step in the process of NO3 " assimilation in plants. Transport is induced by NO3 " and requires both RNA and protein synthesis, leading Jackson, .A. et al . (Plant Phvsiol. 52:120-127 (1973)) to propose that a specific NO3 " transport protein is synthesized. Although much is known about the activity of the NO3 " transporter (Goyal, S.S. et al.. Plant Phvsiol. 82:1051- 1056 (1986)), a NO3 " transport protein has not yet been identified in higher plants.
  • nitrate Once nitrate has been transported into the plant, it is reduced in a multi-enzyme process to ammonia.
  • the reduction of nitrate is closely regulated since nitrate reduction requires substantial energy, and since the final product of the reduction, ammonia, is toxic when accumulated (and must, therefore, itself be transported when present in excess).
  • Nonitrate Reductase The first enzyme in the nitrate reducing pathway is termed “Nitrate Reductase” (or, equivalently, “NR activity”) (Guerrero, M.G. et al.. Ann. Rev. Plant Phvsiol. 32:169-204 (1981)).
  • Plant NR activity is generally thought to be localized in the cytoplasm (Ritenour, G.L., et al .. Plant Phvsiol. 42:233-237 (1966); Dalling, M.J., et al .. Biochim. Biophvs. Acta 283:513-519 (1972); Suzuki, D.A., et al.. Planta 151:457-461 (1981); Oaks, A. et al.. Ann. Rev. Plant Phvsiol. 36:345-365 (1985)).
  • Miflin, B.J. for example, initially found a small percentage of the total barley root NR activity in a mitochondria enriched fraction (Miflin, B.O., Nature 214:1133-1134 (1967)). Miflin, B.J. subsequently reported that a membrane associated NR activity was localized in an unidentified particulate fraction distinct from the mitochondria (Miflin, B.J., Planta 93:160-170 (1970)). Blevins et al . (Plant Phvsiol.
  • Nitrate reductase activity has been found in the cell wall-PM region, and in the tonoplast membranes, of Neurospora crassa (Roldan, J.M., et al.. Plant Phvsiol. 70:872-874 (1982)). Nitrate reductase activity has, however, not been found to be localized in the plasma membrane of higher plants.
  • NR activity Latent nitrate reductase activity was detected in corn (Zea avs L., Golden Jubilee) root microsome fractions.
  • Microsomal-associated NR activity was stimulated up to 20 fold by Triton X-100 (octyl phenoxy polyethoxyethanol) whereas soluble NR activity was only increased up to 1.2 fold.
  • Microsomal-associated NR activity represented up to 19% of the total root NR activity.
  • Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NR activity was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM).
  • the PM associated NR activity was not removed by washing vesicles with up to 1.0 M NaCl but was solubilized from the PM with 0.05% Triton X-100.
  • vanadate sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100.
  • the results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.
  • Membrane associated nitrate reductase was detected in plasma membrane (PM) fractions isolated by aqueous two- phase partitioning from barley (Hordeum vulgare L. var CM 72) roots.
  • the PM associated NR was not removed by washing vesicles with 500 mM NaCl and 1 mM EDTA and represented up to 4% of the total root NR activity.
  • PM associated NR was stimulated up to 20 fold by Triton X-100 whereas soluble NR was only increased 1.7 fold.
  • the latency was a function of the solubilization of NR from the membrane.
  • NR solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chiore!la sorokiniana NR.
  • Anti-NR immunoglobulin G (IgG) fragments purified from the anti-NR serum inhibited NO3 " uptake by more than 90% but had no effect on NO2 " uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum IgG fragments inhibited NO3 " uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO3 " uptake.
  • the invention shows that NO3 " uptake and NO3 " reduction in the PM of barley roots are related.
  • the present invention concerns a "non-cytosolic," “membrane-associated” nitrate reductase activity which is derivable from higher plants.
  • non-cytosolic, membrane-associated nitrate reductase activity of barley, it will be readily perceived that the invention permits the identification and isolation of other non-cytosolic, membrane-associated nitrate reductase activities from other plants, and that such activities, and their uses, are within the scope of the equivalents of the present invention.
  • nitrate reductase or "NR,” as used herein, are intended to refer to enzymes capable of catalyzing the reduction of nitrate. Such enzymes may be referred to either in terms of their physical characteristics (i.e. amino acid sequence, native or denatured molecular weight, etc.), or equivalently, by their functional characteristics (i.e. kinetic constants (Vmax, Km, turnover number, etc.), substrate preferences or specificities, product(s) produced, reaction condition preferences (i.e. thermal, pH, ionic, co-factor, co- enzyme, etc.).
  • activity is intended to refer to and to describe an enzyme by its functional characteristics.
  • an enzyme is "non-cytosolic" if it is normally not present in, or if it is substantially absent from, the cytoplasm of the cell in which it is naturally produced.
  • a non-cytosolic enzyme is one which is, or can be, present in the cytoplasm of the cell in which it is normally and naturally produced.
  • An enzyme is "membrane-associated” if it is bound, or capable of being naturally bound, to the plasma membrane of the cell in which it is naturally produced.
  • a membrane- associated enzyme is one which is, or can be, present in a state bound to the plasma membrane of a cell in which it is normally and naturally produced.
  • a highly enriched source of plasma membranes can be obtained from any of a variety of plant tissue.
  • a preferred source of plasma membrane can be obtained from crude membrane preparations isolated from the roots of corn.
  • Aqueous two- phase partitioning (Larsson, C.H., in Modern Methods of Plant Analysis. N.S. Series, Cell Components, vol. 1, pp. 85-104, Linskens, et al .. eds., Springer-Verlag, Berlin, Heidelberg, New York (1985); Hodges, T.K., and Mills, D., Academic Press i!£:41-54 (1986); Sandstrom, R.P., et al .. Plant Phvsiol. 85:693-698 (1987)) was used to obtain highly enriched PM fractions.
  • a membrane-associated NR activity was localized in the PM.
  • Corn seedlings (Zea mavs L., Golden Jubilee) are preferably grown hydroponically. Seed kernels are preferably treated with 1.5% volume/volume NaOCl prepared from common bleach for 15 min, rinsed with distilled water and germinated at room temperature in aerated distilled water. After approximately 24 h, the germinated seeds are spread on a stainless steel screen about 1 cm above the surface of 51 of an aerated 0.2 M CaU2 solution. The seeds are covered with plastic wrapping and placed in the dark at 25 * C. The plastic wrapping may be removed after 3 d, and after an additional two days, the seedlings are preferably transferred to aerated 0.1- strength Hoagland solution no. 1, lacking nitrogen (Hoagland, D.R.
  • the growth conditions preferably provide 60-65% relative humidity, 25 * C, and continuous light to minimize the effect of rhythms on NR activity levels.
  • the photon flux density at the seedling canopy is approximately 400 as measured at 400- 700 nm with a Licor 190 S sensor and is preferably supplied by incandescent and cool white fluorescent lamps, respectively, at a ratio of 1:5.
  • Lights are preferably obtained from Sylvania Lighting System, Danvers, MA. After 2 d in continuous light the seedlings may be transferred to 0.1 strength Hoagland solution no. 1 containing 1 mM KNO3 for 24 h to induce NR activity.
  • the roots (approximately 25 g) from 9-d-old corn seedlings are excised, preferably just below the seed, and snipped into 1 cm pieces with a pair of scissors.
  • the seedlings are then ground in a suitable amount of cold grinding buffer (i.e. 60 ml) using a chilled mortar and pestle, or other suitable means.
  • a suitable grinding buffer preferably comprises 250 mM sucrose, 3 mM ethylenediamine- tetraacetic acid (EDTA), 50 mM 2-amino-2-(hydroxymethyl)-l,3- propanediol (Tris-HCl) (pH 8.0), 0.05% (w/w roots) polyvinyl- polypyrrolidone, 1 mM dithiothreitol (DTT), 3 M phenylmethylsulfonyl fluoride (PMSF), 1 ⁇ M Na 2 Mo ⁇ 4(H 2 0) , and 5 ⁇ M flavine-adenine dinucleotide (FAD).
  • EDTA ethylenediamine- tetraacetic acid
  • Tris-HCl 2-amino-2-(hydroxymethyl)-l,3- propanediol
  • PMSF phenylmethylsulfonyl fluoride
  • FAD flavine-adenine dinucleotide
  • the PMSF is preferably made up as a 0.1 M stock in isopropanol and added at a ratio of 0.75 ml PMSF: 25 ml grind buffer.
  • the DTT is preferably made up as a 1.0 M stock in deionized water and added at a ratio of 0.025 ml DTT: 25 ml grind buffer. All chemicals used in the grind buffer may be obtained from the Sigma Chemical Co., St. Louis, M0, USA.
  • the homogenate is preferably filtered through 4 layers of cheesecloth and centrifuged preferably at 12,000 g for 10 min.
  • the post- 12,000 g fraction may be saved for enzyme analysis.
  • the supernatant is preferably centrifuged at 120,000 g for 20 min.
  • the resulting pellet represented a microsomal fraction, i.e., a crude membrane fraction.
  • the post-120,000 g fraction may also be used for enzyme analysis.
  • the above-obtained microsomal pellets are preferably suspended in resuspension buffer, diluted to 20 ml, and centrifuged at 120,000 g for 20 min.
  • the resuspension buffer preferably contains 2 mM Tris- HC1 (pH 8.2), 1 ⁇ M Na 2 Mo ⁇ 4(H 2 0)2. 5 ⁇ M FAD, 3 mM PMSF and 1 mM DTT.
  • the process is preferably repeated three more times, and samples from each membrane pellet including the initial icrosome fraction are saved to determine NR activity.
  • Microsomal pellets may alternatively be preferably resuspended in 2.2 ml of 250 mM sucrose, 5 mM K-phosphate (pH 7.8) and 1 mM PMSF, and partitioned in an aqueous polymer two- phase system as detailed by Larsson et al . (Plasma Membranes, In: Modern Methods of Plant Analysis. New Series, Vol. 1, Cell Components, HF Linksens, et al .. eds., Springer-Verlag, Berlin, pp. 85-104 (1985)). All steps in the procedure are preferably carried out at 4'C.
  • a suitable amount of the resuspended microsome fraction (1-5 ml) is preferably added to an approximately 8 ml two- phase system that contained 6.5% (w/w) Dextran T 500 (Pharmacia Fine Chemicals, Piscataway, NJ, USA), 6.5% (w/w) polyethylene glycol 3350 (Sigma Chemical Co., St. Louis, M0, USA), 0.33 M sucrose, 3 mM KC1 and 5 mM K-phosphate (pH 7.8).
  • the contents of the tubes is mixed by inversion 40-50 times, and centrifuged in a swinging-bucket rotor at 1,200 g for 5 min.
  • the upper phase is collected and repartitioned on a fresh lower phase as described by Sandstrom et al . (Plant Phvsiol. 8j>:693-698 (1987)).
  • the remaining microsome (U2) and the first lower phase (L j ) fractions are collected and each is diluted with a suitable amount (such as 20 ml) of resuspension buffer and centrifuged at 120,000 g for 20 min.
  • the U2 pellet is suspended in resuspension buffer, diluted to 20 ml and again centrifuged at 120,000 g for 20 min.
  • the resulting pellets are suspended and diluted to a protein concentration equivalent to the post-120,000 g fraction in resuspension buffer, and may be used directly for enzyme analysis.
  • Twice-centrifuged U2 fractions are diluted to 0.5 mg.ml.j protein concentration in resuspension buffer and brought to a final concentration of 0-1.0 M NaCl in 0.5 ml. All fractions are vigorously mixed for 30 sec and placed on ice. The salt treated fractions were sonicated on ice in, for example, an American Brand sonicator (American Scientific Product, McGraw Park, IL, USA). After 20 min, the fractions are diluted with 8 ml of resuspension buffer and centrifuged at 150,000 g for 15 min. Nitrate reductase activities may be determined in the resuspended pellets.
  • Twice-pelleted U2 fractions may, alternatively, be diluted to 0.5 mg/ml protein with resuspension buffer, and brought to 0 to 0.1% Triton X-100 (octyl phenoxy polyethoxyethanol, Sigma Chemical Co., St. Louis, M0, USA) in a total volume of 0.5 ml.
  • Triton X-100 octyl phenoxy polyethoxyethanol, Sigma Chemical Co., St. Louis, M0, USA
  • the material is then preferably mixed vigorously for approximately 30 s and placed on ice. After 15 min, fractions are centrifuged at 150,000 g for 15 min without dilution. Vanadate-sensitive ATPase activity and NR activity were determined in the resuspended pellets and the soluble fractions.
  • the activities of various enzymes whose use facilitate the present invention may be determined in the following manner.
  • Triton X-100-stimulated nucleoside diphosphatase (UDPase) activity is preferably assayed according to Nagahashi, J. et al . (Protoplasm 112:167-173 (1982)).
  • Triose phosphate isomerase activity is preferably determined according to the method of Gibbs, M. et al .. (Enzymes of glycolysis, In HF Linksens, BD Sanwal, M Tracey, eds., Modern Methods of Plant Analysis, Vol. 7, Springer Verlag Berlin, p. 520, (1964).
  • Cytochrome c oxidase (EC 1.9.3.1.) is preferably assayed according to the method of Hodges, T.K., et al . (Methods Enzvmol 3.2:392-406 (1974)).
  • Antimycin A insensitive NADH Cyt c reductase (EC 1.6.99.3) activity is preferably assayed as described in Lord et al. (J. Cell Biol. 57:659-667 (1973)) and Hodges, T.K. et al. (Methods Enzvmol 32:392-406 (1974)).
  • Vanadate sensitive ATPase (EC 3.6.1.3) activity is determined in the presence and absence of Triton X-100 (Gallagher, S.R., et al.. Plant Phvsiol. 70:1335-1340 (1982); Sandstro , R.P., et al.. Plant Phvsiol. 85:693-698 (1987)).
  • Nitrate sensitive ATPase activity is preferably determined according to O'Neill et al . (Plant Phvsiol. 72:837-846 (1983)).
  • Alcohol dehydrogenase (EC 1.1.1.1) is preferably assayed according to the method of Suzuki, Y. et al . (Phvsiol .
  • Nitrate reductase activity (EC 1.6.6.1) is preferably determined according to the method of Aslam et al . (Plant Phvsiol. 83:579-584 (1987)) in the presence of various concentrations of Triton X-100 (at a known T:p ratio) except that the assay volume was reduced to 500 ⁇ l . All enzyme specific activities are reported on a mg protein basis.
  • Protein concentration is preferably measured using a modification of the Bradford (Bradford, M.M., Anal. Biochem. 71:248-254) method in which 60 ⁇ l of 0.2% Triton X-100 was included in each assay tube to solubilize membrane proteins; crystalline bovine serum albumin (Sigma Chemical Co., St. Louis, M0, USA) was used as a standard.
  • the membrane pellet may be washed, preferably four times, in hypotonic buffer (Pupillo, P. et al .. Planta 151:506-511 (1981)). The initial wash releases approximately 20-25% of the NR activity to the soluble fraction. This probably represents soluble NR activity that was loosely bound or trapped within membrane vesicles. Subsequent washes of the membrane pellet release little additional NR activity. This indicates that NR activity detected in the washed membrane pellets was membrane-associated.
  • the membrane fractions are preferably washed at least two times with hypotonic buffer to remove loosely associated soluble NR activity.
  • This invention further comprises the gene sequences coding for membrae associated nitrate reductase, expression vehicles containing the gene sequence, hosts transformed therewith, the nitrate reductase produced by such transformed host expression, and uses for the nitrate reductase.
  • Any of a variety of procedures may be used to clone the nitrate reductase-encoding gene sequence.
  • One such method entails analyzing a shuttle vector library of cDNA inserts (derived from a nitrate reductase expressing cell) for the presence of an insert which contains the nitrate reductase gene sequence. Such an analysis may be conducted by transfecting cells with the vector and then assaying for NR expression.
  • One method for cloning the nitrate reductase gene sequence entails determining the amino acid sequence of the enzyme molecule. To accomplish this task, NR protein may be purified (using the assays described above), and analyzed to determine the amino acid sequence of the protein. Any method capable of elucidating such a sequence can be employed, however, Edman degradation is preferred. The use of automated sequenators is especially preferred.
  • NR peptide fragments can be obtained by incubating the intact molecule with cyanogen bromide, or with proteases such as papain, chymotrypsin or trypsin (Oike, Y. et al ., J. Biol. Chem. £57:9751-9758 (1982); Liu, C. et al.. Int. J. Pent. Protein Res. 11:209-215 (1983)). If the peptides are greater than 10 amino acids long, the sequence information is generally sufficient to permit one to clone a gene sequence such as that encoding nitrate reductase.
  • the DNA sequences capable of encoding them are examined in order to clone the gene sequence encoding the nitrate reductase. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., In: Molecular Biology of the Gene sequence. 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356-357).
  • amino acid sequence may be encoded by only a single oligonucleotide
  • amino acid sequence may be encoded by any of a set of similar oligonucleotides.
  • all of the members of this set contain oligonucleotides which are capable of encoding the peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene sequence which encodes the peptide fragment
  • only one member of the set contains the nucleotide sequence that is identical to the nucleotide sequence of the gene sequence.
  • this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleo ⁇ tides in the same manner in which one would employ a single oligonucleotide to clone the gene sequence that encodes the peptide.
  • the genetic code Wang, J.D., n: Molecular
  • oligonucleotides can be identified, each of which would be capable of encoding the NR peptides.
  • the probability that a particular oligonucleotide will, in fact, constitute the actual nitrate reductase encoding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells.
  • Such "codon usage rules" are disclosed by Lathe, R., et al .. J. Molec. Biol. 183:1-12 (1985).
  • oligonucleotide or a set of oligonucleotides, that contains a theoretical "most probable" nucleotide sequence capable of encoding the NR peptide sequences is identified.
  • the oligonucleotide, or set of oligonucleotides contain ⁇ ing the theoretical "most probable" sequence capable of encoding the nitrate reductase gene sequence fragments is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probable" sequence, or set of sequences.
  • oligonucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the NR gene sequence (Maniatis, T., et al.. Molecular Cloning A Laboratory Manual . Cold Spring Harbor Press, Cold Spring Harbor, NY (1982).
  • the actual identification of NR peptide sequences permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide.
  • a DNA molecule or set of DNA molecules, capable of functioning as a probe to identify and isolate the NR gene sequence.
  • the process for genetically engineering the nitrate reductase according to the invention is facilitated through the cloning of genetic sequences which are capable of encoding the nitrate reductase and through the expression of such genetic sequences.
  • the term "gene sequence” is intended to refer to a nucleic acid molecule (preferably DNA).
  • Gene sequences which are capable of encoding the nitrate reductase may be derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof.
  • Genomic DNA may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with the 5' promoter region of the NR gene sequences. To the extent that a host cell can recognize the transcriptional regulatory and translational initiation signals associated with the expression of the protein, then the region 5' may be retained and employed for transcriptional and translational initiation regulation.
  • the cDNA may be cloned and the resulting clone screened with an appropriate probe for cDNA coding for the desired sequences.
  • the cDNA may be manipulated in substantially the same manner as the genomic DNA. However, with cDNA there will be no introns or intervening sequences. For this reason, a cDNA molecule which encodes the nitrate reductase is the preferred gene sequence of the present invention.
  • Genomic DNA or cDNA may be obtained in several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from a cell which produces the nitrate reductase and used to produce cDNA by means well known in the art. Such suitable DNA preparations are enzy atically cleaved, or randomly sheared, and ligated into recombinant vectors to form a gene sequence library. Such vectors can then be screened with the above-described oligonucleotide probes in order to identify a NR encoding sequence.
  • a suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the NR gene sequence (or which is complementary to such an oligonucleotide, or set of oligonucleotides) identified using the above-described procedure, is synthesized, and hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation, derived from cells which are capable of expressing the NR gene sequence.
  • the source of DNA or cDNA used will preferably have been enriched for NR sequences. Such enrichment can most easily be obtained from cDNA obtained by extracting RNA from cells which produce high levels of the NR gene sequence.
  • the above-described DNA probe may be labeled with a detectable group.
  • detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of immunoassays and in general most any label useful in such methods can be applied to the present invention.
  • Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem. 12:1243 (1976)), enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes (see U.S. Pat. Nos. 4,230,797 and 4,238,565) and enzyme inhibitors (see U.S. Pat. No. 4,134,792); fluorescers (see Clin. Chem.
  • chromophores such as chemiluminescers and bioluminescers (see Clin. Chem. 15:512 (1979)); specifically bindable ligands; proximal interacting pairs; and radioisotopes such as 3 H, 35 S, 3 P, 125 I and 1 C.
  • labels and labeling pairs are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their reactive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors).
  • a cofactor-labeled probe can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme.
  • an enzyme which acts upon a substrate to generate a product with a measurable physical property.
  • examples of the latter include, but are not limited to, beta-galactosidase, alkaline phosphatase and peroxidase.
  • a library of expression vectors is prepared by cloning DNA or, more preferably cDNA, from a cell capable of expressing NR into an expression vector.
  • the library is then screened for members capable of expressing a protein which binds to anti-NR antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as the NR or fragments of the NR.
  • the cloned nitrate reductase encoding sequences obtained through the methods described above, may be operably linked to an expression vector, and introduced into bacterial, or eukaryotic cells to produce NR, or a functional derivative thereof. Techniques for such manipulations are disclosed by Maniatis, T. et al . , supra, and are well known in the art.
  • the above discussed methods are, therefore, capable of identifying a gene sequence which is capable of encoding the nitrate reductase or fragments thereof.
  • characteristics may include the ability to specifically bind anti-NR antibody, the ability to elicit the production of antibody which are capable of binding to the NR, the ability to provide nitrate reductase activity to a recipient cell, etc.
  • a gene sequence which encodes the NR can be prepared by synthetic means (such as by organic synthetic means, etc.)
  • the nitrate reductase encoding sequences may be operably linked to an expression vector, and introduced into prokaryotic or eukaryotic cells in order to produce the nitrate reductase or its functional derivatives.
  • the present invention pertains both to the intact nitrate reductase and to the functional derivatives of this nitrate reductase.
  • a "functional derivative" of the nitrate reductase is a compound which possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of the nitrate reductase.
  • the term "functional derivative” is intended to include the "fragments,” “variants,” “analogues,” or “chemical derivatives” of a molecule.
  • a “fragment” of a molecule such as the nitrate reductase is meant to refer to any polypeptide subset of the molecule.
  • a “variant” of a molecule such as the nitrate reductase is meant to refer to a molecule substantially similar in structure and function to either the entire mole- cule, or to a fragment thereof.
  • a molecule is said to be “substantially similar” to another molecule if both molecules have substantially similar structures or if both molecules possess a similar biological activity.
  • nitrate reductase an "analog" of a molecule such as the nitrate reductase is meant to refer to a molecule substantially similar in function to either the entire molecule or to a fragment thereof.
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art.
  • a DNA sequence encoding the nitrate reductase or its functional derivatives may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T., et al .. supra, and are well known in the art.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression.
  • regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of the nitrate reductase synthesis.
  • promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of the nitrate reductase synthesis.
  • Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the gene sequence coding for the nitrate reductase may be obtained by the above- described methods.
  • This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation.
  • the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the nitrate reductase gene sequence, or (3) interfere with the ability of the nitrate reductase gene sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the present invention encompasses the expression of the nitrate reductase protein (or a functional derivative thereof) in either prokaryotic or eukaryotic cells.
  • Preferred prokaryotic hosts include bacteria such as E. coli. Bacillus. Streptomvces. Pseudomonas. Salmonella. Serratia. etc.
  • the most preferred prokaryotic host is E. coli.
  • Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E.
  • coli W3110 (F", lambda " , prototrophic (ATCC 27325)), and other enterobacteriu such as Salmonella tvphimurium or Serratia marcescens, and various Pseudomonas species. Under such conditions, the nitrate reductase will not be glycosylated.
  • the procaryotic host must be compatible with the rep!icon and control sequences in the expression plasmid.
  • nitrate reductase or a functional derivative thereof in a prokaryotic cell (such as, for example, E. coli. B. subtilis. Pseudomonas. Streptomvces. etc.)
  • a prokaryotic cell such as, for example, E. coli. B. subtilis. Pseudomonas. Streptomvces. etc.
  • promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
  • constitutive promoters examples include the ijnt promoter of bacteriophage ⁇ , the bla promoter of the actamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, etc.
  • inducible prokaryotic promoters include the major right and left promoters of bacteriophage ⁇ (PL and PR), the trj), recA. lacZ. lad, and flal promoters of E. coli, the ⁇ -amylase (Ulmanen, I., et al .. J. Bacteriol. 162:176-182 (1985)) and the ⁇ -28-specific promoters of B.
  • subtilis (Gil an, M.Z., et al.. Gene sequence 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In: The Molecular Biology of the Bacilli. Academic Press, Inc., NY (1982)), and Streptomvces promoters (Ward, J.M., et L, Mol. Gen. Genet. 103:468-478 (1986)).
  • Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind. M crobiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev.
  • riboso e binding site upstream of the gene sequence-encoding sequence.
  • ribosome binding sites are disclosed, for example, by Gold, L., et al . (Ann. Rev. Microbiol. 35:365-404 (1981)).
  • Preferred eukaryotic hosts include yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture.
  • Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VER0 or CH0-K1, or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or the myeloma P3x63Sg8, and their derivatives.
  • Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332 that may provide better capacities for correct post-translational processing.
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, etc., may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.
  • Yeast provides substantial advantages in that it can also carry out post-translational peptide modifications.
  • Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i.e., pre-peptides).
  • Any of a series of yeast gene sequence expression systems incorporating promoter and termination elements from the actively expressed gene sequences coding for glycolytic enzymes produced in large quantities when yeast are grown in mediums rich in glucose can be utilized.
  • Known glycolytic gene sequences can also provide very efficient transcriptional control signals.
  • the promoter and terminator signals of the phosphoglycerate kinase gene sequence can be utilized.
  • Another preferred host is insect cells, for example the
  • Drosophila larvae Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used. Rubin, G.M., Science 140:1453-1459 (1988).
  • baculovirus vectors can be engineered to express large amounts of the nitrate reductase in insects cells (Jasny, B.R., Science 138:1653 (1987); Miller, D.W., et al .. in Genetic Engineering (1986), Setlow, J.K., et al .. eds., Plenum. Vol. 8, pp. 277-297).
  • eukaryotic regulatory regions Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include the promoter of the mouse etallothionein I gene sequence (Hamer, D., et al .. J Mol. ADD!. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C, et al .. Nature (London.
  • yeast gal4 gene sequence promoter Johnston, S.A., et al.. Proc. Natl . Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P.A., et al.. Proc. Natl. Acad. Sci. (USA) ⁇ 1:5951-5955 (1984)).
  • the presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as the nitrate reductase encoding DNA sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the nitrate reductase encoding sequence).
  • the nitrate reductase encoding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the nitrate reductase may occur through the transient expression of the introduced sequence. Alter ⁇ natively, permanent expression may occur through the integration of the introduced sequence into the host chromosome.
  • a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
  • cDNA expression vectors incorporating such elements include those described by Oka ama, H., Molec. Cell. Biol. 3:280 (1983).
  • the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • a plasmid or viral vector capable of autonomous replication in the recipient host.
  • Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColEl, pSClOl, pACYC 184, ⁇ VX.
  • Such plasmids are, for example, disclosed by Maniatis, T., et al . (In: Molecular Clonin g . A Laboratory Manual . Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)).
  • Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli. Academic Press, NY (1982), pp. 307-329).
  • Suitable Streptomvces plasmids include pIJlOl (Kendall, K.J., et al.. J. Bacteriol.
  • Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives.
  • Such plasmids are well known in the art (Botstein, D., et al .. Miami Wntr. Svmo. 19:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomvces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, J.R., Cell 28:203-204 (1982); Bollon, D.P., et al.. J. Clin. Hematol . Oncol. 10:39- 48 (1980); Maniatis, T., In: Cell Biology: A Comprehensive Treatise. Vol. 3. Gene sequence Expression, Academic Press, NY, pp. 563-608 (1980)).
  • the DNA con- struct(s) may be introduced into an appropriate host cell by any of a variety of suitable means: transformation, transfection, conjugation, protoplast fusion, electro ⁇ poration, calcium phosphate-precipitation, direct microinjection, etc.
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
  • Expression of the cloned gene sequence(s) results in the production of the nitrate reductase, or in the production of a fragment of this nitrate reductase. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).
  • the expressed protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chro atography, affinity chromatography, electrophoresis, or the like.
  • the nitrate reductase gene sequences can be introduced into a plant by genetic engineering techniques, which upon production in the plant cell could be used as a means for facilitating the fixation of nitrogen by the plant, and therby lessen, or obviate, the plants requirement for reduced nitrogen. Therefore, it is possible to produce a plant that is less dependent upon fertilizers, and more able to survive adverse culturing conditions.
  • the NR gene sequence is used to transform a plant to enhance or confer a nitrate reductase activity to the plant.
  • the coding region for an NR gene sequence that may be used in this invention may be the full-length or partial active length of the gene sequence. It is necessary, however, that the genetic sequence coding for the nitrate reductase be expressed, and produced, as a functional nitrate reductase in the resulting plant cell.
  • DNA from both genomic DNA and cDNA and synthetic DNA encoding a nitrate reductase gene sequence may be used in this invention.
  • a nitrate reductase gene sequence may be constructed partially of a cDNA clone, partially of a genomic clone, and partially of a synthetic gene sequence and various combinations thereof.
  • the DNA coding for the nitrate reductase gene sequence may comprise portions from various species.
  • this invention comprises chimeric gene sequences:
  • These additional gene sequences contain sequences for promoter(s) or terminator(s).
  • the plant regulatory sequences may be heterologous or homologous to the host cell.
  • the promoter of the nitrate reductase gene sequence is used to express the chimeric gene sequence.
  • Other promoters that may be used in the gene sequence include nos, ocs, and CaMV promoters.
  • An efficient plant promoter that may be used is an overproducing plant promoter. This promoter in operable linkage with the gene sequence for the nitrate reductase should be capable of promoting expression of then nitrate reductase.
  • Overproduc ⁇ ing plant promoters that may be used in this invention include the promoter of the small subunit (ss) of the ribulose-1,5- biphosphate carboxylase from soybean (Berry-Lowe et al .. «L. Molecular and ADD.
  • the expression of the chimeric gene sequence comprising the nitrate reductase gene sequence is operably linked in correct reading frame with a plant promoter and with a gene sequence secretion signal sequence.
  • the chimeric gene sequence comprising a nitrate reductase gene sequence operably linked to a plant promoter, and in the preferred embodiment with the secretion signal sequences, can be ligated into a suitable cloning vector.
  • plasmid or viral (bacteriophage) vectors containing replication and control sequences derived from species compatible with the host cell are used.
  • the cloning vector will typically carry a replication origin, as well as specific gene sequences that are capable of providing phenotypic selection markers in transformed host cells, typically resistance to antibiotics.
  • the transforming vectors can be selected by these phenotypic markers after transformation in a host cell.
  • Host cells that may be used in this invention include procaryotes, including bacterial hosts such as E. coli. S ⁇ typhimurium. and Serratia arcescens. Eukaryotic hosts such as yeast or filamentous fungi may also be used in this invention.
  • the cloning vector and host cell transformed with the vector are used in this invention typically to increase the copy number of the vector.
  • the vectors containing the nitrate reductase gene sequence can be isolated and, for example, used to introduce the chimeric gene sequences into the plant cells.
  • the genetic material contained in the vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA.
  • the genetic material may also be transferred into the plant cell by using polyethylene glycol which forms a precipitation complex with the genetic material that is taken up by the cell. (Paszkowski et al .. EMBO J. 3:2717-22 (1984)).
  • the nitrate reductase gene sequence may be introduced into the plant cells by electroporation.
  • electroporation fromm et al .. "Expression of Gene sequences Transferred into Monocot and Dicot Plant Cells by Electroporation," Proc. Natl. Acad. Sci. U.S.A. 81:5824 (1985)).
  • plant protoplasts are electro- porated in the presence of plasmids containing the nitrate reductase genetic construct. Electrical impulses of high field strength reversibly permeabilize biome branes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.
  • Selection of the transformed plant cells with the expressed nitrate reductase can be accomplished using the phenotypic markers as described above.
  • Another method of introducing the nitrate reductase gene sequence into plant cells is to infect a plant cell with Agrobacterium tumefaciens transformed with the nitrate reductase gene sequence. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots, roots, and develop further into plants.
  • the nitrate reductase gene sequence can be introduced into appropriate plant cells, for example, by means of the Ti plasmid of Agrobacterium tumefaciens.
  • Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens and is stably integrated into the plant genome. Horsch et al ., "Inheritance of Functional Foreign Genes in Plants," Science 133:496-498 (1984); Fraley et al.. Proc. Natl. Acad. Sci. U.S.A. 80:4803 (1983)).
  • Ti plasmids contain two regions essential for the production of transformed cells. One of these, named transfer DNA (T DNA), induces tumor formation. The other, termed virulent region, is essential for the formation but not maintenance of tumors.
  • the transfer DNA region which transfers to the plant genome, can be increased in size by the insertion of the enzyme's gene sequence without its transferring ability being affected. By removing the tumor- causing gene sequences so that they no longer interfere, the modified Ti plasmid can then be used as a vector for the transfer of the gene sequence constructs of the invention into an appropriate plant cell.
  • All plant cells which can be transformed by Agrobacterium and whole plants regenerated from the transformed cells can also be transformed according to the invention so to produce transformed whole plants which contain the transferred nitrate reductase gene sequence.
  • Method (1) requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts.
  • Method (2) requires (a) that the plant cells or tissues can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants.
  • two plasmids are needed: a T-DNA containing plasmid and a vir plasmid.
  • those plant cells or plants transformed by the Ti plasmid so that the enzyme is expressed can be selected by an appropriate phenotypic marker.
  • phenotypical markers include, but are not limited to, antibiotic resistance.
  • Other phenotypic markers are known in the art and may be used in this invention. All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred nitrate reductase gene sequence.
  • Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrvchis, Trifolium, Trigonella. Vigna. Citrus.
  • Agrobacterium include Ioomoea. Passiflora, Cyclamen, Maius.
  • Regeneration varies from species to species of plants, but generally a suspension of transformed protoplasts containing multiple copies of the nitrate reductase gene sequence is first provided. Embryo formation can then be induced from the protoplast suspensions, to the stage of ripening and germination as natural embryos.
  • the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.
  • the mature plants, grown from the transformed plant cells, are selfed to produce an inbred plant.
  • the inbred plant produces seed containing the gene sequence for the nitrate reductase. These seeds can be grown to produce plants that have the nitrate reductase.
  • the inbreds according to this invention can be used to develop insect tolerant hybrids. In this method, an insect tolerant inbred line is crossed with another inbred line to produce the hybrid. Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are covered by the invention provided that these parts comprise the insect tolerant cells. Progeny and variants, and mutants of the regenerated plants are also included within the scope of this invention.
  • FI first generation hybrids
  • F2 second generation hybrids
  • the genetic distribu ⁇ tion of the F2 hybrids are 1/4 nitrate reductase/nitrate reductase : 1/2 nitrate reductase/wild type : 1/4 wild type/wild type.
  • the F2 hybrids with the genetic makeup of nitrate reductase/nitrate reductase are chosen as the herbicidal tolerant plants.
  • variant describes phenotypic changes that are stable and heritable, including heritable variation that is sexually transmitted to progeny of plants, provided that the variant still comprises an insect tolerant plant.
  • mutant describes variation as a result of environmental conditions, such as radiation, or as a result of genetic variation in which a trait is transmitted meiotically according to well-established laws of inheritance. The mutant plant, however, must still exhibit an insect tolerance according to the invention.
  • Corn seedlings (Zea mays L., Golden Jubilee) were grown hydroponically. Seeds (kernels) were soaked in 1.5% volume/volume NaOCl prepared from common bleach for 15 min, rinsed with distilled water and germinated at room temperature in aerated distilled water. After 24 h, the germinated seeds were spread on a stainless steel screen about 1 cm above the surface of 5 1 of aerated 0.2 mM CaCl2 solution, covered with plastic wrapping and placed in the dark at 25'C. The plastic wrapping was removed after 3 d. After 5 d the seedlings were transferred to aerated 0.1- strength Hoagland solution no.
  • Roots (25 g) from 9-d-old corn seedlings were excised just below the seed, snipped into 1 cm pieces with a pair of scissors, and then ground in 60 ml of cold grinding buffer in a chilled mortar and pestle.
  • the grinding buffer consisted of 250 mM sucrose, 3 mM ethylenediaminetetraacetic acid (EDTA), 50 mM 2-amino-2- (hydroxymethyl)-l,3-propanediol (Tris-HCl) (pH 8.0), 0.05% (w/w roots) polyvinylpolypyrrolidone, 1 mM dithiothreitol (DTT), 3 mM phenylmethylsulfonyl fluoride (PMSF), 1 ⁇ M Na2 o ⁇ 4(H2 ⁇ )2 » and 5 ⁇ M flavine-adenine dinucleotide (FAD).
  • EDTA ethylenediaminetetraacetic acid
  • Tris-HCl 2-amino-2- (hydroxymethyl)-l,3-propanediol
  • PMSF phenylmethylsulfonyl fluoride
  • FAD flavine-adenine dinucleotide
  • the PMSF was made up as a 0.1 M stock in isopropanol and added at a ratio of 0.75 ml PMSF: 25 ml grind buffer.
  • the DTT was made up as a 1.0 M stock in deionized water and added at a ratio of 0.025 ml DTT: 25 ml grind buffer. All chemicals used in the grind buffer were obtained from Sigma Chemical Co., St. Louis, M0, USA.
  • the homogenate was filtered through 4 layers of cheesecloth and centrifuged at 12,000.g for 10 min. In some experiments a portion of the post-12,000.g fraction was saved for enzyme analysis. The supernatant was centrifuged at 120,000.g for 20 min.
  • the resulting pellet represented a microsomal fraction, i.e., a crude membrane fraction.
  • the post-120,000.g fraction was used in some experiments for enzyme analysis.
  • microsomal pellets were suspended in resuspension buffer, diluted to 20 ml, and centrifuged at 120,000.g for 20 min.
  • the resuspension buffer was 2 mM Tris-HCl (pH 8.2), 1 ⁇ M Na 2 Mo0 4 (H 2 0)2, 5 ⁇ M FAD, 3 mM PMSF and 1 mM DTT. The process was repeated three more times, and samples from each membrane pellet including the initial microsome fraction were saved to determine NRA.
  • Plasma-membrane isolation Plasma-membrane isolation. Microsomal pellets were resuspended in 2.2 ml of 250 mM sucrose, 5 mM K-phosphate (pH 7.8) and 1 mM PMSF, and partitioned in an aqueous polymer two- phase system as detailed by Larsson et al . (Plasma Membranes, In: Modern Methods of Plant Analysis. New Series, Vol. 1, Cell Components, HF Linksens, et al .. eds., Springer-Verlag, Berlin, pp. 85-104 (1985)). All steps in the procedure were carried out at 4 * C.
  • Nitrate reductase activity was detected in a crude membrane fraction isolated from corn roots.
  • the membrane pellet was washed four times in hypotonic buffer (Pupillo, P., and Del Grosso, E., Planta 151:506-511 (1981)).
  • the initial wash released 23% of the NRA to the soluble fraction. This probably represented soluble NRA that was loosely bound or trapped within membrane vesicles.
  • Subsequent washes of the membrane pellet released little additional NRA. This indicated that NRA detected in the washed membrane pellets was membrane-associated.
  • the membrane fractions were washed at least two times with hypotonic buffer to remove loosely associated soluble NRA.
  • Triton X-100 The effect of Triton X-100 on both soluble and membrane- associated NR activity is shown in Table 1. Microsomal NR activity was stimulated up to nine fold by Triton X-100 while soluble NR activity was hardly affected. Maximum activity was reached at a Triton X-100 concentration of 0.043% which represented a T:p ratio of approx. 3.5 mg.mg.j. Higher Triton X-100 concentrations had little additional effect on the microsomal NR activity. Since the detergent activation was much greater for the membrane-associated NR activity than for the soluble NR activity, the stimulation by Triton X-100 was not the consequence of a general effect on NR activity but appeared to be a specific activation of the membrane- associated enzyme.
  • particulate cellulase is stimulated by Triton X-100 while soluble cellulase is little affected (Koehler, D.E., et al.. Plant Phvsiol. 58:324-330 (1976)).
  • Aqueous two-phase partitioning was used to isolate a PM fraction of very high purity from corn roots (Larsson, C.H., in Modern Methods of Plant Analysis, N.S. Series, Cell Components, vol. 1, pp. 85-104, Linskens, et al . , eds., Springer-Verlag, Berlin, Heidelberg, New York (1985); Hodges, T.K., and Mills, D., Academic Press 118:41-54 (1986)). Marker enzyme assessment of aqueous two-phase partitioned corn-root microsome fractions is shown in Table 2. A 2.3 fold enrichment of the PM H + -ATPase activity was detected in the U2 fraction.
  • corn root fractions were isolated as described above. Preparations of the above fractions were used immediately after isolation to preserve activity and it was not possible to isolate a sufficient amount of the PM fraction to complete all the assays at one time; hence, several different preparations were required to complete all of the assays. Each of the assays was repeated at least three times using three different preparations providing the various fractions. The means ⁇ SE are shown.
  • Triton X-100 stimulation of membrane-associated NR activity indicates that the NADH oxidation sites of the PM NR activity and - or NO3 " reducing sites were not accessible to NADH and - or NO3 " .
  • NADH dependent ferricyanide reductase activity associated with corn root PM fractions was also stimulated six-fold by 0.025% Triton X-100, leading Buckhout and Hrubec (Buckhout, T.J., and Hrubec, T.C., Protoplas a 135:144-154 (1986)) to suggest that the primary site of NAD(P)H oxidation and - or ferricyanide reduction was not exposed to the outside surface of the PM.
  • Singer Singer (Singer, S.J., Annu. Rev. Biochem. 43:805-833 (1974)) proposed that detergent activation was characteristic of enzymes which are deeply buried in a membrane.
  • Nitrate Reductase Activity Specific (nmol.mg protein.h "1 )
  • Table 3 because the results are based on the protein concentration of the reaction mixture.
  • the protein analysis included the membranes, whereas in Table 4, the membranes were separated from the preparation by centrifugation after NR activity was solubilized by the Triton X-100 treatment. The concentration of protein in the supernatant solution was then determined.
  • Plasma membrane NR activity was induced by 1 mM NO3 " above a very low control level which was present in PM fractions isolated from roots of plants rigorously kept free of NO3 " . This low amount of NR activity may be constitutive or the result of a low level of NO3 " contamination which we are unable to detect in the system.
  • Membrane NR activity was unstable; time dependent decreases in PM NR activity occurred when the PM fractions were kept at 0 * C in the presence of protease inhibitors.
  • Plasma membrane NR activity solubilized with Triton X-100 and the soluble NR activity from corn roots were inactivated by antiserum prepared to NR activity purified from barley leaves.
  • the anti-NR activity serum (Somers, D.A., et al.. Plant Phvsiol. 72:949-952 (1983)) was kindly supplied to us by Dr. R. L. Warner, Washington State University, Pullman, WA.
  • latent NR activity was identified in corn-root PM fractions isolated by aqueous two-phase partitioning.
  • the NR activity was not removed from PM vesicles by washing with increasing NaCl concentration up to 1 M but it was solubilized by Triton X-100 (0.05%).
  • Triton X-100 0.05%). The results show that an integral membrane protein capable of reducing nitrate is present in the PM of corn roots.
  • the antiserum cross-reacted with soluble NR from barley roots as evidenced by inactivation of NR activity).
  • the inhibitory effect was only partially reversible; uptake recovered to 50% of the control after 3 rinses in fresh uptake solution and a 10 min equilibration period.
  • IgG fragments isolated from preimmune serum inhibited nitrate uptake 36% but the effect was completely reversible by rinsing.
  • Intact anti-NR molecules did not affect NO3 " uptake (Table 5) presumably because they are much larger (150 kD) than the cleaved fragments (50 kD) and could not move as easily through the root cell wall to bind to NO3 " transport sites.
  • Intact antibodies to the yeast PM Pi binding protein did not affect in vivo Pi uptake until the yeast cell wall was removed or the IgG molecules were cleaved with papain. This lead Jeanjean et al. (Jeanjean, R. et al .. Arch. Microbiol. 137:215-219 (1984)) to suggest that the intact IgG molecules were too large to penetrate the yeast cell wall to bind to the Pi transporter.
  • Triton X-100 also activates the PM ATPase possibly by altering the lipid environment near the ATPase possibly by altering the lipid environment near the ATPase or by removing an inhibitory component (Sandstrom, R.P. et al.. Plant Phvsiol. ⁇ 5:693-698 (1987)). Evaluation of markers for the tonoplast, Golgi apparatus, mitochondria, ER, plastid and cytoplasm demonstrated that each of these activities was significantly reduced by the two-phase procedure.
  • NR is generally considered to be a cytosolic enzyme (Oaks, A. et al .. Ann. Rev. PI Phvsiol. 36:345-365 (1985)).
  • the marker enzyme data indicate that the upper phase fraction consisted mainly of PM.
  • NR activity distribution in root soluble and phase partitioned membrane fractions is shown in Table 7. Most of the NR activity was soluble as is well established (Oaks, A. et al.. Ann. Rev. PI Phvsiol. 36:345-365 (1985)); however, approximately 4% of the total NR was associated with the membrane fraction when assayed in the presence of Triton. A membrane fraction when assayed in the presence of Triton. A 1.8 to 5.4 fold enrichment of the NR activity in the upper phase (PM) fraction over the microsome in the presence and absence of Triton, respectively, (Table 7) was detected. Repeated resuspension and repelleting of the PM fractions failed to decrease NR activity.
  • PM upper phase
  • Triton X-100 Triton X-100. The experiment was repeated 5 times data from a representative experiment are shown.
  • the PM associated NR was inactivated by NR antiserum but was not affected by the preimmune serum.
  • a single spot on Western blots of Triton solubilized PM fractions separated by two- dimensional electrophoresis was detected with the NR antiserum. preparation).
  • the inactivation of the PM associated NR and the inhibition of NO3 " transport by the NR antiserum present the possibility that NO3 " transport and the PM associated NR may be related.
  • the PM associated NR may be a part of an enzyme complex that both transports and reduces NO3 " as proposed by Butz and Jackson (Butz, R.G. et al .. Phvtochem. 16:409-417 (1977)).
  • the NO3 " transporter and the PM associated NR may be completely separate but antigenically related systems.
  • Rufty et al . (Rufty, T.W., Jr. et al .. Plant Phvsiol . ⁇ 1:675-680 (1986)) recently propose the possibility that the receptor for the NR induction system and the functional NR protein may both be associated with the PM of root cells.
  • nitrate reductase was solubilize from the PM fractions as described above.
  • One hundred ⁇ l of water (- antiserum), preimmune serum, or anti-NR antiserum was added to 200 ⁇ l of the Triton solubilized PM fractions.
  • the fractions were kept on ice for 2 h and then centrifuged for 5 min at 12,000 RPM in a microfuge.
  • Nitrate reductase was assayed in the supernatant fractions. Each experiment was repeated at least 4 times and representative date are shown.

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Abstract

Membrane associée à l'activité de réductase du nitrate des plantes utile pour stimuler ou pour donner la capacité de réduire l'azote à une plante ou à une cellule végétale. Une molécule recombinante, et une cellule transformée capable d'exprimer une telle activité de réductase du nitrate sont décrites.
PCT/US1990/004397 1989-09-15 1990-08-06 Proteine associee a la membrane de plantes, qui possede une activite de reductase du nitrate WO1991004325A1 (fr)

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EP (1) EP0491780A4 (fr)
JP (1) JPH05500156A (fr)
AU (1) AU639026B2 (fr)
CA (1) CA2065873A1 (fr)
NZ (1) NZ234941A (fr)
WO (1) WO1991004325A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2688228A1 (fr) * 1992-03-05 1993-09-10 Agronomique Inst Nat Rech Procede pour accroitre la precocite d'une plante et/ou abaisser la teneur en nitrates stockes dans la plante.
WO1997030163A1 (fr) * 1996-02-14 1997-08-21 The Governors Of The University Of Alberta Plantes capables d'une assimilation/metabolisation amelioree de l'azote
US7390937B2 (en) 1996-02-14 2008-06-24 The Governors Of The University Of Alberta Plants with enhanced levels of nitrogen utilization proteins in their root epidermis and uses thereof
US7560626B2 (en) 2005-12-23 2009-07-14 Arcadia Biosciences, Inc. Promoter sequence obtained from rice and methods of use
US7786343B2 (en) 1996-02-14 2010-08-31 The Governors Of The University Of Alberta Transgenic plants expressing recombinant barley alanine aminotransferase
US8288611B2 (en) 2005-12-23 2012-10-16 Arcadia Biosciences, Inc. Nitrogen-efficient monocot plants
CN112941036A (zh) * 2021-02-08 2021-06-11 吉林迈丰生物药业有限公司 一种提高狂犬病毒在人二倍体细胞中复制水平的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01132382A (ja) * 1985-10-28 1989-05-24 General Hospital Corp:The 硝酸レダクタ−ゼについてコ−ドするポリヌクレオチド

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01132382A (ja) * 1985-10-28 1989-05-24 General Hospital Corp:The 硝酸レダクタ−ゼについてコ−ドするポリヌクレオチド

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Biochemica et Biophysica Acta, Volume 708, issued 1982, T.M. KUO et. al., "NADH-Nitrate Reductase in Barley Leaves Identification and Amino Acid Composition of Sub Unit Protein", see pages 75-81. *
Eur. J. Biochemistry, Volume 158, issued 1986, CRASKE et. al., "The Respiratory Nitrate Reductase from Paracoccus denitrifica pr s," see page 429-436. *
FEMS Microbiology Letters, Volume 11, issued 1981, C.G. BARR et. al., "Cloning the chl C Gene for Nitrate Reductase of Escherichia coli, pages 213-216, see entire document. *
Gene, Volume 85, No. 2, issued 1989, F. DANIEL-VEDELE, "Cloning and Analysis of the Tomato Nitrate Reductase-Encoding Gene: Protein Domain Structure and Amino Acid Homologies in Higher Plants", see pages 371-380. *
NATO Advanced Science Institute Series: Series A, Volume 61, Genetic Engineering in Eucaryotes, issued 1983, A. KLEINHOFS et al., "Nitrate Reductase Genes as Selectable Markers for Plant Cell Transformation", see pages 215-231. *
See also references of EP0491780A4 *
The EMBO Journal, Volume 7, No. No. 11, issued 1988, C-L. CHENG et al., "A New Locus (NIAI) in Arabidopsis thaliana encoding Nitrate Reductase", see pages 3309-3314. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2688228A1 (fr) * 1992-03-05 1993-09-10 Agronomique Inst Nat Rech Procede pour accroitre la precocite d'une plante et/ou abaisser la teneur en nitrates stockes dans la plante.
WO1993018154A3 (fr) * 1992-03-05 1993-10-28 Agronomique Inst Nat Rech Procede pour accroitre la precocite d'une plante et/ou abaisser la teneur en nitrates stockes dans la plante
US5569833A (en) * 1992-03-05 1996-10-29 Institut National De La Recherche Agronomique Method for enhancing the earliness of a plant and/or lowering the content of nitrates stored in the plant
US7390937B2 (en) 1996-02-14 2008-06-24 The Governors Of The University Of Alberta Plants with enhanced levels of nitrogen utilization proteins in their root epidermis and uses thereof
GB2325232A (en) * 1996-02-14 1998-11-18 Univ Alberta Plants having enhanced nitrogen assimilation/metabolism
GB2325232B (en) * 1996-02-14 2000-11-29 Univ Alberta Plants having enhanced nitrogen assimilation/metabolism
WO1997030163A1 (fr) * 1996-02-14 1997-08-21 The Governors Of The University Of Alberta Plantes capables d'une assimilation/metabolisation amelioree de l'azote
US7786343B2 (en) 1996-02-14 2010-08-31 The Governors Of The University Of Alberta Transgenic plants expressing recombinant barley alanine aminotransferase
US8115062B2 (en) 1996-02-14 2012-02-14 The Governors Of The University Of Alberta Transgenic plants expressing recombinant barley alanine aminotransferase
US7560626B2 (en) 2005-12-23 2009-07-14 Arcadia Biosciences, Inc. Promoter sequence obtained from rice and methods of use
US7982093B2 (en) 2005-12-23 2011-07-19 Arcadia Biosciences, Inc. Promoter sequence obtained from rice and methods of use
US8288611B2 (en) 2005-12-23 2012-10-16 Arcadia Biosciences, Inc. Nitrogen-efficient monocot plants
US8642840B2 (en) 2005-12-23 2014-02-04 Arcadia Biosciences, Inc. Nitrogen-efficient monocot plants
CN112941036A (zh) * 2021-02-08 2021-06-11 吉林迈丰生物药业有限公司 一种提高狂犬病毒在人二倍体细胞中复制水平的方法

Also Published As

Publication number Publication date
AU6359890A (en) 1991-04-18
NZ234941A (en) 1992-06-25
AU639026B2 (en) 1993-07-15
EP0491780A4 (en) 1992-11-19
EP0491780A1 (fr) 1992-07-01
CA2065873A1 (fr) 1991-03-16
JPH05500156A (ja) 1993-01-21

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