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WO2011106734A1 - Plants de riz et de soja transgéniques dotés de caractéristiques de croissance améliorées - Google Patents

Plants de riz et de soja transgéniques dotés de caractéristiques de croissance améliorées Download PDF

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Publication number
WO2011106734A1
WO2011106734A1 PCT/US2011/026368 US2011026368W WO2011106734A1 WO 2011106734 A1 WO2011106734 A1 WO 2011106734A1 US 2011026368 W US2011026368 W US 2011026368W WO 2011106734 A1 WO2011106734 A1 WO 2011106734A1
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Prior art keywords
seq
plant
gpt
plants
transgenic
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Pat J. Unkefer
Penelope S. Anderson
Thomas J. Knight
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University of Maine System
Los Alamos National Security LLC
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University of Maine System
Los Alamos National Security LLC
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Priority claimed from US12/660,506 external-priority patent/US20110030104A1/en
Priority claimed from US12/660,501 external-priority patent/US20110030089A1/en
Priority claimed from US12/660,508 external-priority patent/US20110004961A1/en
Application filed by University of Maine System, Los Alamos National Security LLC filed Critical University of Maine System
Publication of WO2011106734A1 publication Critical patent/WO2011106734A1/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
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • 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
    • 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

  • Soybean and rice are field crops of particular importance: Soybean ⁇ Glycine max) is the world's leading source of vegetable oil and protein meal, however, soybeans are also used for the production of milk, flour, tofu, curd, and ice cream. The oil extracted from soybeans is used for cooking oil, margarine, and salad dressings. Industrial uses for processed soybean oil include ingredients for paints, plastics, fibers, detergents, cosmetics, and lubricants. Soybean is also used as a food source for both animals and humans. Soybean is widely used as a source of protein for animal feeds for poultry, swine and cattle. For human
  • soybean meal is made into soybean flour which is processed to protein concentrates used for meat extenders or specialty pet foods. Production of edible protein ingredients from soybean offers a healthy, less expensive replacement for animal protein in meats as well as dairy-type products.
  • Rice ⁇ Oryza sativa L. is one of the principle food crops of the world.
  • the annual world wide rice production output is around 390 million metric tons.
  • Rice is a simple grain crop that is abundant in starch content.
  • Rice is an ancient crop that has been a staple food supply for thousands of years.
  • Rice has also been processed into products as diverse as cereals, vinegar, and wine (sake). It is of extreme economic and cultural value to many countries. Especially in developing countries rice has persisted as a principle dietary component until today.
  • the success of crop plants is dependent on their ability to maximize growth and yield outputs. Plant growth and biomass accumulation are dependent on the availability, absorption and assimilation of nutrients from the environment. Nitrogen is one of the principal factors limiting vegetative production. Only a few
  • microorganisms are capable of reducing molecular atmospheric nitrogen to a form usable by the plant. Plants themselves are unable to do this. The majority of plants obtain nitrogen by assimilating nitrate and ammonium from the soil into the nitrogen carriers glutamine and glutamate. Glutamine and glutamate are the donors for the biosynthesis of major nitrogen-containing compounds, including amino acids, nucleotides, chlorophylls, polyamines, and alkaloids (Miflin and Lea, 1980, In Miflin B J (ed) The Biochemistry of Plants Vol 5 pp. 169-202, Academic Press Inc., London).
  • Nitrogen sources are often the major components in fertilizers (Hageman and Lambert, 1 . Corn and Corn Improvement, 3rd ed., Sprague & Dudley, American Society of Agronomy, pp. 431 - 461 , 1988). All field crops have a fundamental dependence on exogenous nitrogen sources. Nitrogenous fertilizer, which is usually supplied as ammonium nitrate, potassium nitrate or urea, typically accounts for 40% of the costs associated with crops in intensive agriculture.
  • MMt metric tons
  • nitrogenous fertilizers Despite the heavy use of nitrogenous fertilizers in agricultural settings, it is estimated that 50% to 70% of the applied nitrogen is lost from the plant-soil system. Because nitrate is soluble and not retained by the soil matrix, excess nitrate may leach into the water and be depleted by microorganisms. In fact, most of the applied nitrogen is rapidly depleted by soil microorganisms, leaching, and other factors, rather than being taken up by the plants. Additionally, use of nitrogenous fertilizers has a plethora of detrimental environmental effects, which are manifested in increased eutrophication, acid rain, soil acidification, and the greenhouse effect. To decrease the agricultural dependence on nitrogenous fertilizers, it would be beneficial to increase nitrogen-use efficiency in plants.
  • Increasing nitrogen use efficiency would enable the production of higher yields with existing fertilizer inputs, enable existing crop yields to be obtained with lower fertilizer input or enable better yields from soils of poorer quality (Good et al. (2004) Trends Plant Sci. 9:57-605). Higher amounts of proteins in the crops can also be produced more cost-effectively.
  • a reduction in fertilizer application would also lessen the environmental damage resulting from extensive nitrogenous fertilizer use.
  • Increased nitrogen utilization efficiency by plants would have a number of other beneficial effects. For example, nitrogen utilization efficient plants would be able to grow and yield better than conventional plants in nitrogen poor soils. The use of nitrogen efficient plants would reduce the requirement for the addition of nitrogenous fertilizers to crops. Since fertilizer application accounts for a significant percentage of the costs associated with crop production, such a reduction in fertilizer use would result in a direct monetary savings.
  • transgenic plant technologies may be gradually increasing, particularly in the United States, Canada and Australia, many regions of the World remain slow to adopt genetically modified plants in agriculture, notably Europe. Therefore, consonant with pursuing the objectives of responsible and sustainable agriculture, there is a strong interest in the development of genetically engineered plants that do not introduce toxins or other potentially problematic substances into plants and/or the environment. There is also a strong interest in minimizing the cost of achieving objectives such as improving herbicide tolerance, pest and disease resistance, and overall crop yields. Accordingly, there remains a need for transgenic plants that can meet these objectives.
  • ribulose bisphosphate carboxylase catalyzes the combination of CO 2 with ribulose bisphosphate to produce 3-phosphoglycerate, a three carbon compound (C-3) that the plant uses to synthesize carbon-containing compounds.
  • CO 2 is combined with phosphoenol pyruvate to form acids containing four carbons (C-4), in a reaction catalyzed by the enzyme phosphoenol pyruvate carboxylase.
  • the acids are transferred to bundle sheath cells, where they are decarboxylated to release CO 2 , which is then combined with ribulose bisphosphate in the same reaction employed by C-3 plants.
  • GS glutamine synthetase
  • GS also reassimilates ammonia released as a result of photorespiration and the breakdown of proteins and nitrogen transport compounds.
  • GS enzymes may be divided into two general classes, one representing the cytoplasmic form (GS1 ) and the other representing the plastidic (i.e., chloroplastic) form (GS2).
  • transgenic tobacco plants overexpressing the full length Alfalfa GS1 coding sequence contained greatly elevated levels of GS transcript, and GS polypeptide which assembled into active enzyme, but did not report phenotypic effects on growth (Temple et al., 1993, Molecular and General Genetics 236: 315-325).
  • Corruzi et al. have reported that transgenic tobacco overexpressing a pea cytosolic GS1 transgene under the control of the CaMV S35 promoter show increased GS activity, increased cytosolic GS protein, and improved growth characteristics (U.S. Patent No. 6,107,547). Unkefer et al.
  • transgenic tobacco plants overexpressing the Alfalfa GS1 in foliar tissues which had been screened for increased leaf-to-root GS activity following genetic segregation by selfing to achieve increased GS1 transgene copy number, were found to produce increased 2-hydroxy-5-oxoproline levels in their foliar portions, which was found to lead to markedly increased growth rates over wildtype tobacco plants (see, U.S. Patent Nos. 6,555,500; 6,593,275; and 6,831 ,040).
  • Unkefer et al. have further described the use of 2-hydroxy-5-oxoproline (also known as 2-oxoglutaramate) to improve plant growth (U.S. Patent Nos. 6,555,500; 6,593,275; 6,831 ,040).
  • 2-hydroxy-5-oxoproline also known as 2-oxoglutaramate
  • Unkefer et al. disclose that increased concentrations of 2-hydroxy-5-oxoproline in foliar tissues (relative to root tissues) triggers a cascade of events that result in increased plant growth characteristics.
  • the invention relates to transgenic soybean and rice plants exhibiting enhanced growth rates, seed and fruit yields, and overall biomass yields, as well as methods for generating growth-enhanced transgenic soybean and rice plants.
  • the invention further relates to soybean and rice glutamine phenylpyruvate transaminase (GPT) proteins, nucleic acid molecules encoding soybean and rice GPT proteins, and uses thereof.
  • GPT glutamine phenylpyruvate transaminase
  • transgenic soybean and rice plants are provided that are engineered to over-express both glutamine phenylpyruvate transaminase (GPT) and glutamine synthetase (GS).
  • GPT glutamine phenylpyruvate transaminase
  • GS glutamine synthetase
  • the GPT+GS double-transgenic plants of the invention exhibit enhanced growth characteristics, with TO generation lines showing an increase in relative growth rate of 67% over wild type soybean plants.
  • Biochemical differences between transgenic and wild type soybean plants were also shown, including a 7-fold increase in leaf GPT activity, a doubling of leaf GS activity, and an 8-fold increase in 2-oxoglutaramate concentration in leaf tissue.
  • GPT glutamine phenylpyruvate transaminase
  • This aspect of the invention is exemplified herein by the disclosure of GPT polynucleotides encoding GPTs from Rice, Soybean, and several species, including Arabidopsis, Grape, Barley, bamboo and a non-plant homolog from Zebra fish, most of which have been expressed as recombinant GPTs and confirmed as having GPT activity.
  • the invention further provides transgenic plants which either express a GPT transgene or both a GPT transgene and a GS transgene.
  • the transgenic plants of the invention are capable of producing higher overall yields over shorter periods of time, and therefore may provide agricultural industries with enhanced productivity across a wide range of crops.
  • the invention utilizes natural plant genes encoding a natural plant enzyme.
  • the enhanced growth characteristics of the transgenic plants of the invention is achieved essentially by introducing either additional GPT capacity or both additional GPT and GS capacity into the plant.
  • the transgenic plants of the invention do not express any toxic substances, growth hormones, viral or bacterial gene products, and are therefore free of many of the concerns that have heretofore impeded the adoption of transgenic plants in certain parts of the World.
  • the invention provides a transgenic plant including a GPT transgene, wherein said GPT transgene is operably linked to a plant promoter.
  • the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 46 and SEQ ID NO: 49, and (b) an amino acid sequence that is at least 75% identical to any one of SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 46 and SEQ ID NO:
  • the GPT transgene construct is incorporated into the genome of a plant selected from the group consisting of: soybean, rice, maize, sugar cane, wheat, oats, sorghum, switch grass, tubers (such as potatoes), canola, lupins or cotton.
  • the invention provides a transgenic plant including a GPT transgene and a GS transgene, wherein said GPT transgene and said GS transgene are operably linked to a plant promoter.
  • the GS transgene is a GS1 transgene.
  • the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 46 and SEQ ID NO: 49, and (b) an amino acid sequence that is at least 75% identical to any one of SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 46 and SEQ ID NO: 49, and (b
  • the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 4, SEQ ID NO: 7 from residue 1 1 and SEQ ID NO: 41 , and (b) an amino acid sequence that is at least 75% identical to SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 41 .
  • the GPT and GS transgenes are incorporated into the genome of the plant.
  • the GPT transgene and a GS transgene construct is incorporated into the genome of a plant selected from the group consisting of: soybean, rice, maize, sugar cane, wheat, oats, sorghum, switch grass, tubers (such as potatoes), canola, lupins or cotton.
  • the invention also provides progeny of any generation of the transgenic plants of the invention, wherein said progeny includes either a GPT transgene or a both a GPT transgene and a GS transgene, as well as a seed of any generation of the transgenic plants of the invention, wherein said seed includes either said GPT transgene or said both GPT transgene and said GS transgene.
  • the transgenic plants of the invention may display one or more enhanced growth characteristics when compared to an analogous wild-type or untransformed plant, including without limitation increased relative growth rate, biomass yield, seed yield, flower or flower bud yield, fruit or pod yield, larger leaves, and may also display increased levels of GPT and/or GS activity, and/or increased levels of 2-oxoglutaramate.
  • the transgenic plants of the invention display increased nitrogen use efficiency or increased tolerance to salt or saline conditions.
  • transplastomic plant or cell line carrying a either a GPT transgene expression cassette or a GPT and a GS transgene expression cassette, said expression cassette being flanked by sequences from the plant or plant cell's plastome.
  • the invention provides a method for preparing a transplastomic plant or cell line carrying a GPT transgene and a GS transgene construct, said method including the steps of: (a) inserting into at least one expression cassette at least a GPT transgene and a GS transgene, wherein said expression cassette is flanked by sequences from the plant or plant cell's plastome.
  • Methods for producing the transgenic plants of the invention and seeds thereof are also provided, including methods for producing a plant having enhanced growth properties, increased nitrogen use efficiency and increased tolerance to germination or growth in salt or saline conditions, relative to an analogous wild type or untransformed plant.
  • the invention provides isolated nucleic acid molecules encoding GPT.
  • Exemplary GPT polynucleotides and GPT polypeptides are provided herein.
  • the invention provides isolated GPT polynucleotides encoding rice GPTs. Included are GPT polynucleotides encoding the GPT proteins of SEQ ID NO: 1 1 and SEQ ID NO: 32. Specific embodiments include the GPT polynucleotide sequence of SEQ ID NO:10, as well as polynucleotides encoding the GPT amino acid sequences of SEQ ID NO: 1 1 and SEQ ID NO: 32.
  • the invention provides isolated GPT polynucleotides encoding soybean GPTs. Included are GPT polynucleotides encoding the GPT proteins SEQ ID NO: 13, SEQ ID NO: 33 and SEQ ID NO: 33 with a further Isoleucine at the N-terminus of the sequence. Specific embodiments include the GPT polynucleotide sequence of SEQ ID NO: 12, as well as polynucleotides encoding the GPT amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 33 and SEQ ID NO: 33 with a further Isoleucine at the N-terminus of the sequence.
  • the invention provides an isolated GPT polynucleotide having a sequence selected from the group consisting of (a) the nucleotide sequence of SEQ ID NO: 1 ; (b) a nucleotide sequence having at least 75% identity to SEQ ID NO: 1 , and encoding a polypeptide having GPT activity; (c) a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, or a polypeptide having at least 75% sequence identity thereto which has GPT activity; and, (d) a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 truncated at its amino terminus by between 30 to 56 amino acid residues, or a polypeptide having at least 75% sequence identity thereto which has GPT activity.
  • the isolated GPT polynucleotide includes the nucleotide sequence of SEQ ID NO: 18, SEQ ID NO: 29, SEQ ID NO: 45 or SEQ ID NO: 48, or a nucleotide sequence having at least 75% identity to SEQ ID NO: 18, SEQ ID NO: 29, SEQ ID NO: 45 or SEQ ID NO: 48.
  • the invention provides an isolated GPT polynucleotide encoding a polypeptide having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 46 and SEQ ID NO: 49, and (b) an amino acid sequence that is at least 75% identical to any one of SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO
  • the invention provides a nucleic acid construct including a plant promoter operably linked to a GPT polynucleotide.
  • the plant promoter is a heterologous promoter.
  • the plant promoter is a heterologous tissue-specific promoter.
  • Related aspects include a vector including such a nucleic acid construct, and a host cell including such a vector or nucleic acid construct.
  • the host cell is a plant cell.
  • the host cell is a plant cell which expresses the GPT polynucleotide.
  • the host cell is a plant cell which expresses the GPT polynucleotide, wherein polynucleotide so expressed has GPT activity.
  • the invention further provides a plant organ, embryo or seed including such a nucleic acid construct or vector, wherein the plant organ, embryo or seed expresses the GPT polynucleotide.
  • the GPT polynucleotide expressed has GPT activity.
  • the invention provides a transgenic plant including such a nucleic acid construct or vector, wherein the transgenic plant expresses the polynucleotide, which in one embodiment has GPT activity.
  • Progeny and seed of such a transgenic plant, wherein the progeny or seed includes the GPT polynucleotide are also provided.
  • FIG. 1 Nitrogen assimilation and 2-oxoglutaramate biosynthesis: schematic of metabolic pathway.
  • FIG. 2. Multiple sequence alignment of the amino acid sequences of several putative plant, algal and animal GPT proteins, showing a high degree of structural idenity and conservation (dark grey shading indicates amino acid residues which are identical in all sequences aligned, and light grey shading indicates amino acids that are identical in all but one or two sequences aligned).
  • This alignment compares (in order from top to bottom in each block) the plant GPTs from barley ⁇ Hordeum vulgare; SEQ ID NO: 46), rice ⁇ Orzya sativa residues 2 to 468; SEQ ID NO: 1 1 ) , corn ⁇ Zea mays; SEQ ID NO: 52), cotton ⁇ Gossypium hirsutum; SEQ ID NO: 53), grape ⁇ Vitis vinifera residues 1 1 to 459; SEQ ID NO: 9), castor oil plant (Ricinus communis; SEQ ID NO: 54), California poplar (Populus trichocarpa; SEQ ID NO: 55), soybean [Glycine max; SEQ ID NO: 56), Zebra fish ⁇ Danio rerio; SEQ ID NO: 35), arabidopsis ⁇ Arabidopsis thaliana; SEQ ID NO: 2), a Bryophyte moss ⁇ Physcomitrella patens; SEQ ID
  • the consensus sequence is SEQ ID NO: 59.
  • the alignment includes the presumed amino-terminal targeting sequence, if known.
  • FIG. 3. Subset of the multiple sequence alignment of the of FIG. 2, showing a very high degree of structural identity and conservation (dark grey shading indicates amino acid residues which are identical in all sequences aligned, and light grey shading indicates amino acids that are identical in all but one or two sequences aligned).
  • This alignment includes all sequences aligned and displayed in FIG. 2, except the Physcomitrella and Chlamydomonas sequences. As will be appreciated, relative to the alignment of FIG.
  • This alignment compares (in order from top to bottom in each block) the plant GPTs from cotton ⁇ Gossypium hirsutum; SEQ ID NO: 53), grape ⁇ Vitis vinifera residues 1 1 to 459; SEQ ID NO: 9), castor oil plant ⁇ Ricinus communis; SEQ ID NO: 54), California poplar ⁇ Populus trichocarpa; SEQ ID NO: 55), soybean ⁇ Glycine max; SEQ ID NO: 56), Zebra fish ⁇ Danio rerio; SEQ ID NO: 35), arabidopsis ⁇ Arabidopsis thaliana; SEQ ID NO: 2), barley ⁇ Hordeum vulgare; SEQ ID NO: 46), rice ⁇ Orzya sativa residues 2 to 468; SEQ ID NO: 1 1 ), and corn ⁇ Zea mays; SEQ ID NO: 52).
  • the consensus sequence is SEQ ID NO: 60).
  • FIG. 4 Photograph showing comparison of transgenic tobacco plants over- expressing either GS1 or GPT, compared to wild type tobacco plant. From left to right: wild type plant, Alfalfa GS1 transgene, Arabidopsis GPT transgene. See Examples 3 and 5, infra.
  • FIG. 5 Photograph showing comparison of transgenic Micro-Tom tomato plants over-expressing either GS1 or GPT, compared to wild type tomato plant. From left to right: wild type plant, Alfalfa GS1 transgene, Arabidopsis GPT transgene. See Examples 4 and 6, infra.
  • FIG. 6 Photographs showing comparisons of leaf sizes between wild type and GS1 or GPT transgenic tobacco plants.
  • A Comparison between leaves from GS1 transgenic tobacco (bottom leaf) and wild type (top leaf).
  • B Comparison between leaves from GPT transgenic tobacco (bottom leaf) and wild type (top leaf).
  • FIG. 7 Photographs showing comparisons of transgenic tobacco plants generated from various crosses between GS1 and GPT transgenic tobacco lines with wild type and single transgene plants.
  • A-C Cross 2, 3 and 7, respectively. See Example 7, infra.
  • A Comparison between leaves from GSXGPT Cross 3 (bottom leaf) and wild type (top leaf).
  • B Comparison between leaves from GSXGPT Cross 7 (bottom leaf) and wild type (top leaf). See Example 7, infra.
  • FIG. 9. Photograph of transgenic pepper plant (right) and wild type control pepper plant (left), showing larger pepper fruit yield in the transgenic plant relative to the wild type control plant. See Example 8, infra.
  • FIG. 1 1 Photograph of transgenic bean plant (right) and wild type control bean plant (left), showing increased growth in the transgenic plant relative to the wild type control plant.
  • FIG. 12 Transgenic bean plants pods, flowers and flower buds compared to wild type control bean plants (transgenic line expressing grape GPT and Arabidopsis GS transgenes). See Example 10, infra.
  • FIG. 13 Photograph of transgenic bean plant (right) and wild type control bean plant (left), showing increased growth in the transgenic plant relative to the wild type control plant.
  • Transgenic line expressing Grape GPT and Arabidopsis GS transgenes See Example 10, infra.
  • FIG. 14 Transgenic Cowpea Line A plants compared to wild type control Cowpea plants (transgenic line expressing Arabidopsis GPT and GS transgenes), showing that the transgenic plants grow faster and flower and set pods sooner than wild type control plants.
  • FIG. 15 Photograph of transgenic Cowpea Line A plant (right) and wild type control Cowpea plant (left), showing increased growth in the transgenic plant relative to the wild type control plant.
  • FIG. 16 Transgenic Cowpea Line G plants compared to wild type control Cowpea plants (transgenic line expressing Grape GPT and Arabidopsis GS transgenes), showing that the transgenic plants grow faster and flower and set pods sooner than wild type control plants.
  • A plant heights
  • B flowers and pea pod numbers
  • C leaf bud and trifolate numbers. See Example 12, infra.
  • FIG. 17 Photograph of transgenic Cowpea Line G plant (right) and wild type control Cowpea plant (left), showing increased growth in the transgenic plant relative to the wild type control plant.
  • FIG. 18 Photograph of transgenic Cantaloupe plant (right) and wild type control Cantaloupe plant (left), showing increased growth in the transgenic plant relative to the wild type control plant.
  • FIG. 19 Photograph of transgenic Pumpkin plants (right) and wild type control Pumpkin plants (left), showing increased growth in the transgenic plants relative to the wild type control plants.
  • FIG. 20 Photograph of transgenic Arabidopsis plants (right) and wild type control Arabidopsis plants (left), showing increased growth in the transgenic plants relative to the wild type control plants.
  • Transgenic lines expressing Arabidopsis GPT and GS transgenes See Example 16, infra.
  • FIG. 21 Transgenic tomato plants expressing Arabidopsis GPT and GS transgenes compared to control tomato plants.
  • A Photograph of transgenic tomato plant leaves (right) vs. wild type control leaves (left) showing larger leaves in the transgenic plant.
  • B Photograph of transgenic tomato plants (right) and wild type control plants (left), showing increased growth in the transgenic plants relative to the wild type control plants. See Example 17, infra.
  • FIG. 22 Photograph of transgenic Camelina plant (right) and wild type control Camelina plant (left), showing increased growth in the transgenic plant relative to the wild type control plant.
  • FIG. 23 Photograph of transgenic rice plant (right) and wild type control rice plant (left), showing increased growth in the transgenic plant relative to the wild type plant.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof ("polynucleotides”) in either single- or double-stranded form.
  • polynucleotide encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof ⁇ e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991 , Nucleic Acid Res. 19: 5081 ; Ohtsuka et al., 1985 J. Biol. Chem. 260: 2605-2608; and Cassol et al., 1992; Rossolini et al., 1994, Mol. Cell. Probes 8: 91 - 98).
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • promoter refers to anucleic acid control sequence or sequences that direct transcription of an operably linked nucleic acid.
  • a "plant promoter” is a promoter that functions in plants. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter that is active under environmental or developmental regulation.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • a nucleic acid expression control sequence such as a promoter, or array of transcription factor binding sites
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups ⁇ e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • plant includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, reproductive organs, embryos and parts thereof, etc.), seedlings, seeds and plant cells and progeny thereof.
  • the class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous.
  • GPT polynucleotide and “GPT nucleic acid” are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a polypeptide involved in catalyzing the synthesis of 2- oxoglutaramate, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof.
  • GPT coding sequence refers to the part of the gene which is transcribed and encodes a GPT protein.
  • targeting sequence refers to the amino terminal part of a protein which directs the protein into a subcellular compartment of a cell, such as a chloroplast in a plant cell.
  • GPT polynucleotides are further defined by their ability to hybridize under defined conditions to the GPT polynucleotides specifically disclosed herein, or to PCR products derived therefrom.
  • a "GPT transgene” is a nucleic acid molecule including a GPT polynucleotide which is exogenous to transgenic plant, or plant embryo, organ or seed, harboring the nucleic acid molecule, or which is exogenous to an ancestor plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the GPT polynucleotide.
  • the exogenous GPT transgene will be heterogeneous with any GPT polynucleotide sequence present in wild-type plant, or plant embryo, organ or seed into which the GPT transgene is inserted.
  • the scope of the heterogeneity required need only be a single nucleotide difference.
  • the heterogeneity will be in the order of an identity between sequences selected from the following identities: 0.01 %, 0.05%, 0.1 %, 0.5%, 1 %, 5%, 10%, 15%, and 20%.
  • GS polynucleotide and “GS nucleic acid” are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a glutamine synthetase protein, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof.
  • GS coding sequence refers to the part of the gene which is transcribed and encodes a GS protein.
  • GS1 polynucleotide and “GS1 nucleic acid” are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a glutamine synthetase isoform 1 protein, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof.
  • GS1 coding sequence refers to the part of the gene which is transcribed and encodes a GS1 protein.
  • a "GS transgene” is a nucleic acid molecule including a GS polynucleotide which is exogenous to transgenic plant, or plant embryo, organ or seed, harboring the nucleic acid molecule, or which is exogenous to an ancestor plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the GS polynucleotide.
  • a "GS1 transgene” is a nucleic acid molecule including a GS1 polynucleotide which is exogenous to transgenic plant, or plant embryo, organ or seed, harboring the nucleic acid molecule, or which is exogenous to an ancestor plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the GS1 polynucleotide. More particularly, the exogenous GS or GS1 transgene will be heterogeneous with any GS or GS1 polynucleotide sequence present in wild-type plant, or plant embryo, organ or seed into which the GS or GS1 transgene is inserted.
  • heterogeneity required need only be a single nucleotide difference.
  • heterogeneity will be in the order of an identity between sequences selected from the following identities: 0.01 %, 0.05%, 0.1 %, 0.5%, 1 %, 5%, 10%, 15%, and 20%.
  • GPT polynucleotides of the invention include GPT coding sequences for Arabidopsis, Rice, Barley, bamboo, Soybean, Grape, Clementine orange and Zebra Fish GPTs.
  • Partial length GPT polynucleotides include polynucleotide sequences encoding N- or C-terminal truncations of GPT, mature GPT (without targeting sequence) as well as sequences encoding domains of GPT.
  • Exemplary GPT polynucleotides encoding N- terminal truncations of GPT include Arabidopsis -30, -45 and -56 constructs, in which coding sequences for the first 30, 45, and 56, respectively, amino acids of the full length GPT structure of SEQ ID NO: 2 are eliminated.
  • the inserted polynucleotide sequence need not be identical, but may be only "substantially identical" to a sequence of the gene from which it was derived, as further defined below.
  • the term "GPT polynucleotide” specifically encompasses such substantially identical variants.
  • a number of polynucleotide sequences will encode the same polypeptide, and all such polynucleotide sequences are meant to be included in the term GPT polynucleotide.
  • the term specifically includes those sequences substantially identical (determined as described below) with an GPT polynucleotide sequence disclosed herein and that encode polypeptides that are either mutants of wild type GPT polypeptides or retain the function of the GPT polypeptide (e.g., resulting from conservative substitutions of amino acids in a GPT polypeptide).
  • the term “GPT polynucleotide” therefore also includes such substantially identical variants.
  • the term “conservatively modified variants” applies to both amino acid and nucleic acid sequences.
  • conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1 ) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) ⁇ see, e.g., Creighton, Proteins (1 984)).
  • Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et ai, Molecular Biology of the Cell (3 rd ed., 1 994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1 980).
  • Primary structure refers to the amino acid sequence of a particular peptide.
  • “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long.
  • Typical domains are made up of sections of lesser organization such as stretches of ⁇ -sheet and cc- helices.
  • Tetiary structure refers to the complete three dimensional structure of a polypeptide monomer.
  • Quaternary structure refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
  • isolated refers to material which is substantially or essentially free from components which normally accompany the material as it is found in its native or natural state. However, the term “isolated” is not intended refer to the components present in an electrophoretic gel or other separation medium. An isolated component is free from such separation media and in a form ready for use in another application or already in use in the new application/milieu.
  • An "isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the antibody will be purified (1 ) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N- terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid includes two or more subsequences that are not found in the same relationship to each other in nature.
  • a nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a nucleic acid encoding a protein from one source and a nucleic acid encoding a peptide sequence from another source.
  • a heterologous protein indicates that the protein includes two or more subsequences that are not found in the same relationship to each other in nature ⁇ e.g., a fusion protein).
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms, or by manual alignment and visual inspection.
  • This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
  • This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
  • percentage of sequence identity is used in reference to polypeptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the polypeptide. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981 , Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol.
  • BLAST and BLAST 2.0 are used, typically with the default parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences ⁇ see, e.g., Karlin & Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01 , and most preferably less than about 0.001 .
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, highly stringent conditions are selected to be about 5-10°C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.
  • Tm thermal melting point
  • Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1 .0M sodium ion, typically about 0.01 to 1 .0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Genomic DNA or cDNA including GPT polynucleotides may be identified in standard Southern blots under stringent conditions using the GPT polynucleotide sequences disclosed here.
  • suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1 M NaCI, 1 % SDS at 37°C, and at least one wash in 0.2 X SSC at a temperature of at least about 50°C, usually about 55°C to about 60°C, for 20 minutes, or equivalent conditions.
  • a positive hybridization is at least twice background.
  • alternative hybridization and wash conditions may be utilized to provide conditions of similar stringency.
  • a further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot.
  • GLUTAMINE PHENYLPYRUVATE TRANSAMINASE (GPT) TRANSGENE The present invention discloses for the first time that plants contain a glutamine phenylpyruvate transaminase (GPT) enzyme which is directly functional in the synthesis of the signal metabolite 2-hydroxy-5-oxoproline (2-oxoglutaramate).
  • GPT glutamine phenylpyruvate transaminase
  • 2- Oxoglutaramate is a powerful signal metabolite which regulates the function of a large number of genes involved in the photosynthesis apparatus, carbon fixation and nitrogen metabolism.
  • GPT polynucleotide coding sequences derived from several plant and animal species, and have successfully incorporated the gene into heterologous transgenic host plants which exhibit markedly improved growth characteristics, including faster growth, higher foliar protein content, increased glutamine synthetase activity in foliar tissue, and faster CO 2 fixation rates. It is expected that all plant species contain a GPT which functions in the same metabolic pathway, involving the biosynthesis of the signal metabolite 2-hydroxy-5- oxoproline. Thus, in the practice of the invention, any plant gene encoding a GPT homolog or functional variants thereof may be useful in the generation of transgenic plants of this invention.
  • GPT glutamine phenylpyruvate transaminase
  • the invention provides transgenic plants, and plant organs, embryos and seeds including GPT polynucleotides, which are expressed therein, as well as progeny thereof.
  • GPT polynucleotides which are expressed therein, as well as progeny thereof.
  • GPT proteins and GPT gene coding sequences isolated from a number of plant species. As disclosed herein, GPT proteins share remarkable structural similarity within plant species, and are active in catalyzing the synthesis of 2-oxoglutaramate.
  • the invention provides the sequences of various GPT polynucleotides encoding GPT proteins, as well as the sequences of various GPT polypeptides which may be encoded by GPT polynucleotides, including GPTs derived from Soybean, Rice, Arabidopsis, Grape, Barley, bamboo and a non-plant homolog from Zebra fish, all but one of which (Bamboo) have been expressed as recombinant GPTs and confirmed as having GPT activity.
  • GPTs derived from Soybean, Rice, Arabidopsis, Grape, Barley, bamboo and a non-plant homolog from Zebra fish all but one of which (Bamboo) have been expressed as recombinant GPTs and confirmed as having GPT activity.
  • GPT polynucleotide constructs in which all or part of the coding sequence of the GPT targeting sequence have been deleted have been expressed in transgenic plants and/or in E. coli to establish that the encoded GPT protein is expressed as an active G
  • GPT polynucleotide and protein sequences disclosed herein include without limitation those derived from cotton, castor, poplar, moss and algae, all of which show significant to high structural identity and homology to the aforementioned GPT protein sequences.
  • FIG. 2 is a multiple sequence alignment of the amino acid sequences of several putative plant, algal and animal GPT proteins, showing a high degree of structural idenity and conservation.
  • a high degree of structural conservation is seen beginning at alignment residue 90, likely at or near the amino- terminus of a mature GPT protein following proteolytic cleavage of the target sequence (sequence beginning with VAKR in all but two sequences)
  • little structural homology is seen in the presumed targeting sequences. With respect to the plant sequences, this may be a consequence of the natural variability in chloroplast targeting sequences among different plants.
  • the first ten of these aligned sequences terminate (C-terminus) at alignment residue position 473-475.
  • GPT polynucleotides encoding Rice GPTs are provided, and include GPT polynucleotides encoding the GPT proteins of SEQ ID NO: 1 1 and SEQ ID NO: 32. Specific embodiments include the GPT polynucleotide sequence of SEQ ID NO:10, as well as polynucleotides encoding the GPT amino acid sequences of SEQ ID NO: 1 1 and SEQ ID NO: 32.
  • GPT polynucleotides encoding Soybean GPTs are provided, and include GPT polynucleotides encoding the GPT proteins SEQ ID NO: 13, SEQ ID NO: 33 and SEQ ID NO: 33 with a further Isoleucine at the N-terminus of the sequence.
  • Specific embodiments include the GPT polynucleotide sequence of SEQ ID NO: 12, as well as polynucleotides encoding the GPT amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 33 and SEQ ID NO: 33 with a further Isoleucine at the N-terminus of the sequence.
  • GPT polynucleotides encoding Arabidopsis GPTs are provided, and include GPT polynucleotides encoding the GPT proteins of SEQ ID NO: 2, SEQ ID NO: 21 and SEQ ID NO: 30. Specific embodiments include the GPT polynucleotide sequence of SEQ ID NO: 1 , SEQ ID NO: 18 and SEQ ID NO: 20, as well as polynucleotides encoding the GPT amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 29.
  • GPT polynucleotides encoding Grape GPTs are provided, and include GPT polynucleotides encoding the GPT proteins of SEQ ID NO: 9 and SEQ ID NO: 31 .
  • Specific embodiments include the GPT polynucleotide sequence of SEQ ID NO: 8, as well as polynucleotides encoding the GPT amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 31 .
  • GPT polynucleotides encoding Barley GPTs are provided, and include GPT polynucleotides encoding the GPT proteins of SEQ ID NO: 15 and SEQ ID NO: 34.
  • GPT polynucleotide sequence of SEQ ID NO: 14 as well as polynucleotides encoding the GPT amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 34.
  • GPT polynucleotides Zebra fish Rice GPTs are provided, and include GPT polynucleotides encoding the GPT proteins of SEQ ID NO: 17 and SEQ ID NO: 35.
  • Specific embodiments include the GPT polynucleotide sequence of SEQ ID NO: 16, as well as polynucleotides encoding the GPT amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 35.
  • GPT polynucleotides encoding bamboo GPTs are provided, and include GPT polynucleotides encoding the GPT proteins of SEQ ID NO: 36. Specific embodiments include a GPT polynucleotide sequence encoding the GPT amino acid sequence of SEQ ID NO: 36.
  • GPT polynucleotides encoding Clementine GPTs are provided, and include GPT polynucleotides encoding the GPT proteins of SEQ ID NO: 49. Specific embodiments include a GPT polynucleotide sequence encoding the GPT amino acid sequence of SEQ ID NO: 49.
  • oligonucleotide probes based on the sequences of the GPT polynucleotides disclosed herein can be used to identify the desired gene in a cDNA or genomic DNA library.
  • genomic libraries large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector.
  • cDNA library To prepare a cDNA library, mRNA is isolated from the desired organ, such as ovules, and a cDNA library which contains the GPT gene transcript is prepared from the mRNA. Alternatively, cDNA may be prepared from mRNA extracted from other tissues in which GPT genes or homologs are expressed. cDNA or genomic libraries may be screened using a probe based upon the sequence of a GPT polynucleotide disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Alternatively, antibodies raised against an GPT polypeptide can be used to screen an mRNA expression library.
  • GPT polynucleotides may also be amplified from nucleic acid samples using nucleic acid amplification techniques, such as polymerase chain reaction (PCR), which may be used to amplify the sequences of GPT genes directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other amplification methods may also be useful, for example, to clone GPT polynucleotide encoding GPT proteins for expression, prepare transgene constructs and expression vectors, generate transgenic plants, make oligonucleotide probes for detecting the presence of GPT mRNA in samples, for nucleic acid sequencing, or for other purposes.
  • PCR Protocols A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990).
  • Appropriate primers and probes for identifying GPT polynucleotides from plant tissues may be generated from the GPT polynucleotide sequences provided herein. Alignments of one or more of the GPT polynucleotides (genes) disclosed herein, and/or alignments of one or more of the GPT protein amino acid sequences disclosed herein, may be used to identify conserved regions in the GPT structure suitable for preparing the appropriate primer and probe sequences. Primers that specifically hybridize to conserved regions in one of the plant GPT polynucleotides disclosed herein may be used to amplify sequences from widely divergent plant species.
  • GPT polynucleotides may be tested for their ability to direct the expression of a functional, biologically active GPT protein by expressing the GPT polynucleotide in a cell and assaying for GPT activity or the presence of increased levels of 2- oxoglutaramate. Assays for GPT activity and 2-oxogltaramate are disclosed herein (see Examples). In addition, GPT polypeptides may be tested in transgenic plants, following protocols in the Examples which follow. Plants expressing a GPT transgene will show increased levels of GPT activity, higher levels of 2- oxoglutaramate, and/or enhanced growth characteristics, relative to wild type plants (see Examples following).
  • the GPT polynucleotides are useful in directing the expression of recombinant GPT polypeptides in recombinant expression systems, as is generally known.
  • the GPT polynucleotides are useful in generating transgenic plants with increased levels of GPT activity, upregulated 2-oxoglutaramate levels, and enhanced growth characteristics.
  • numerous species of transgenic plants containing a GPT transgene showed enhanced growth characteristics, including increased biomass, earlier and more productive flowering, increased fruit or pod yields, larger leaf sizes, taller heights, tolerance to high salt germination and faster growth.
  • the gene coding sequence for the desired transgene(s) must be incorporated into a nucleic acid construct (also interchangeably referred to herein as a/an (transgene) expression vector, expression cassette, expression construct or expressible genetic construct), which can direct the expression of the transgene sequence in transformed plant cells.
  • a nucleic acid construct also interchangeably referred to herein as a/an (transgene) expression vector, expression cassette, expression construct or expressible genetic construct
  • Such nucleic acid constructs carrying the transgene(s) of interest may be introduced into a plant cell or cells using a number of methods known in the art, including but not limited to electroporation, DNA bombardment or biolistic approaches, microinjection, and via the use of various DNA-based vectors such as Agrobacterium tumefaciens and Agrobacterium rhizogenes vectors.
  • the nucleic acid construct may direct the expression of the incorporated transgene(s) (i.e., GPT), either in a transient or stable fashion.
  • Stable expression is preferred, and is achieved by utilizing plant transformation vectors which are able to direct the chromosomal integration of the transgene construct.
  • the coding sequence thereof in E. coli or another suitable host and determine whether the 2-oxoglutaramate signal metabolite is synthesized at increased levels (see Examples 19-23). Where such an increase is demonstrated, the coding sequence may then be introduced into both homologous plant hosts and heterologous plant hosts, and growth characteristics evaluated. Any assay that is capable of detecting 2-oxoglutaramate with specificity may be used for this purpose, including without limitation the NMR and HPLC assays described in Example 2, infra. In addition, assays which measure GPT activity directly may be employed, such as the GPT activity assay described in Example 7.
  • a typical plant expression vector for generating transgenic plants will comprise the transgene of interest under the expression regulatory control of a promoter, a selectable marker for assisting in the selection of transformants, and a transcriptional terminator sequence.
  • the basic elements of a nucleic acid construct for use in generating the transgenic plants of the invention are: a suitable promoter capable of directing the functional expression of the transgene(s) in a transformed plant cell, the transgene (s) (i.e., GPT coding sequence) operably linked to the promoter, preferably a suitable transcription termination sequence (i.e., nopaline synthetic enzyme gene terminator) operably linked to the transgene, and sometimes other elements useful for controlling the expression of the transgene, as well as one or more selectable marker genes suitable for selecting the desired transgenic product (i.e., antibiotic resistance genes).
  • a suitable promoter capable of directing the functional expression of the transgene(s) in a transformed plant cell
  • the transgene (s) i.e., GPT coding sequence
  • a suitable transcription termination sequence i.e., nopaline synthetic enzyme gene terminator
  • polynucleotides may be used to generate the transgenic plants of the invention.
  • GPT proteins are highly conserved among various plant species, and it is evident from the experimental data disclosed herein that closely-related non-plant GPTs may be used as well (e.g., Danio rerio GPT). Therefore, various plant GPT genes may be used to generate growth-enhanced transgenic plants in a variety of heterologous plant species.
  • GPT transgenes expressed in a homologous plant would be expected to result in the desired enhanced-growth characteristics as well (i.e., rice glutamine transaminase over-expressed in transgenic rice plants), although it is possible that regulation within a homologous cell may attenuate the expression of the transgene in some fashion that may not be operable in a heterologous cell.
  • GPT polynucleotides suitable for use as GPT transgenes in the practice of the invention may be obtained by various means, as will be appreciated by one skilled in the art, tested for the ability to direct the expression of a GPT with GPT activity in a recombinant expression system, i.e., E. coli (see Examples 20-23), in a transient in planta expression system (see Example 19), or in a transgenic plant (see Examples 1 -18).
  • the GPT gene functions as one of at least two transgenes incorporated into the transgenic plants of the invention, the other being the glutamine sythetase gene (see infra).
  • GS glutamine synthetase
  • GPT glutamine synthetase
  • Glutamine synthetase plays a key role in nitrogen metabolism in plants, as well as in animals and bacteria.
  • the GS enzyme catalyzes the addition of ammonium to glutamate to synthesize glutamine in an ATP-dependent reaction.
  • GS enzymes from assorted species show highly conserved amino acid residues considered to be important for active site function, indicating that GS enzymes function similarly (for review, see Eisenberg et al., Biochimica et Biophysica Acta, 1477:122 145, 2000).
  • GS is distributed in different subcellular locations (chloroplast and cytoplasm) and is found in various plant tissues, including leaf, root, shoot, seeds and fruits.
  • cystolic isoform GS1
  • plastidic chloroplastic isoform
  • GS2 is principally found in leaf tissue and functions in the assimilation of ammonia produced by photorespiration or by nitrate reduction.
  • GS1 is mainly found in leaf and root tissue, typically exists in a number of different isoforms in higher plants, and functions to assimilate ammonia produced by all other physiological processes (Coruzzi, 1991 , Plant Science 74: 145-155; McGrath and Coruzzi, 1991 , Plant J. 1 (3): 275-280; Lam et al., 1996, Ann. Rev. Plant Physiol. Plant Mol. Biol. 47: 569-593; Stitt, 1999, Curr. Op. Plant Biol. 2: 178-186; Oliveira et al., 2001 , Brazilian J. Med. Biol. Res. 34: 567-575).
  • Multiple GS genes are associated with a complex promoter repertoire which enable the expression of GS in an organ and tissue specific manner, as well as in an environmental factor- dependent manner.
  • Plant glutamine synthetase consists of eight subunits, and the native enzyme in plants has a molecular mass ranging from 320 to 380 kD, each subunit having a molecular mass of between 38 and 45 kD.
  • Chloroplastic GS2 appears to be encoded by a single gene, while various cystoloic GS1 isoforms are encoded within multigene families (Tingey et al., 1987, supra; Sakamoto et al., 1989, Plant Mol. Biol. 13: 61 1 -614; Brears et al, 1991 , supra; Li et al., 1993, Plant Mol. Biol., 23:401 -407; Dubois et al., 1996, Plant Mol. Biol., 31 :803- 817; Lam et al., 1996, supra).
  • GS1 multigene families appear to encode different subunits which may combine to form homo- or hetero-octamers, and the different members show a unique expression pattern suggesting that the gene members are differentially regulated, which may relate to the various functional roles of glutamine synthetase plays in overall nitrogen metabolism (Gebhardt et al., 1986, supra; Tingey et al., 1987, supra; Bennett et al., 1989, supra; Walker and Coruzzi, 1989, supra; Peterman and Goodman, 1991 , Mol Gen Genet. 1991 ;330:145-154.; Marsolier et al., 1995, supra; Temple et al., 1995, supra; Dubois et al., 1996, supra).
  • a GS1 gene coding sequence is employed to generate GS transgene constructs.
  • the Alfalfa or Arabidopsis GS1 gene coding sequence is used to generate a transgene construct that may be used to generate a transgenic plant expressing the GS1 transgene.
  • a construct may be used to transform Agrobacteria.
  • the transformed Agrobacteria are then used to generate T 0 transgenic plants.
  • Example 5 demonstrates the generation of T 0 GS1 transgenic tobacco plants using this approach.
  • Examples 6 and 17 demonstrates the generation of T 0 GS1 transgenic tomato plants
  • Example 8 demonstrates the generation of T 0 GS1 transgenic pepper plants
  • Examples 9 and 10 demonstrate the generation of T 0 GS1 transgenic bean plants
  • Examples 1 1 and 12 demonstrate the generation of T 0 GS1 transgenic cowpea plants
  • Example 13 demonstrates the generation of T 0 GS1 transgenic alfalfa plants
  • Example 14 demonstrates the generation of T 0 GS1 transgenic cantaloupe plants
  • Example 15 demonstrates the generation of T 0 GS1 transgenic pumpkin plants
  • Example 16 demonstrates the generation of T 0 GS1 transgenic Arabidopsis plants
  • Example 18 demonstrates the generation of T 0 GS1 transgenic Cantaloupe.
  • the gene coding sequence for the desired transgene(s) must be incorporated into a nucleic acid construct (also interchangeably referred to herein as a/an (transgene) expression vector, expression cassette, expression construct or expressible genetic construct), which can direct the expression of the transgene sequence in transformed plant cells.
  • a nucleic acid construct also interchangeably referred to herein as a/an (transgene) expression vector, expression cassette, expression construct or expressible genetic construct
  • Such nucleic acid constructs carrying the transgene(s) of interest may be introduced into a plant cell or cells using a number of methods known in the art, including but not limited to electroporation, DNA bombardment or biolistic approaches, microinjection, and via the use of various DNA-based vectors such as Agrobacterium tumefaciens and Agrobacterium rhizogenes vectors.
  • the nucleic acid construct may direct the expression of the incorporated transgene(s) (i.e., GPT), either in a transient or stable fashion.
  • Stable expression is preferred, and is achieved by utilizing plant transformation vectors which are able to direct the chromosomal integration of the transgene construct.
  • a typical plant expression vector for generating transgenic plants will comprise the transgene of interest under the expression regulatory control of a promoter, a selectable marker for assisting in the selection of transformants, and a transcriptional terminator sequence.
  • the basic elements of a nucleic acid construct for use in generating the transgenic plants of the invention are: a suitable promoter capable of directing the functional expression of the transgene(s) in a transformed plant cell, the transgene (s) (i.e., GPT coding sequence) operably linked to the promoter, preferably a suitable transcription termination sequence (i.e., nopaline synthetic enzyme gene terminator) operably linked to the transgene, and sometimes other elements useful for controlling the expression of the transgene, as well as one or more selectable marker genes suitable for selecting the desired transgenic product (i.e., antibiotic resistance genes).
  • a suitable promoter capable of directing the functional expression of the transgene(s) in a transformed plant cell
  • the transgene (s) i.e., GPT coding sequence
  • a suitable transcription termination sequence i.e., nopaline synthetic enzyme gene terminator
  • Agrobacterium tumefaciens is the primary transformation system used to generate transgenic plants, there are numerous vectors designed for Agrobacterium transformation.
  • Agrobacterium systems utilize "binary" vectors that permit plasmid manipulation in both E. coli and Agrobacterium, and typically contain one or more selectable markers to recover transformed plants (Hellens et al., 2000, Technical focus: A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446-451 ).
  • Binary vectors for use in Agrobacterium transformation systems typically comprise the borders of T-DNA, multiple cloning sites, replication functions for Escherichia coli and A. tumefaciens, and selectable marker and reporter genes.
  • So-called "super-binary" vectors provide higher transformation efficiencies, and generally comprise additional virulence genes from a Ti (Komari et al., 2006, Methods Mol. Biol. 343: 15-41 ).
  • Super binary vectors are typically used in plants which exhibit lower transformation efficiencies, such as cereals.
  • Such additional virulence genes include without limitation virB, virE, and virG (Vain et al., 2004, The effect of additional virulence genes on transformation efficiency, transgene integration and expression in rice plants using the pGreen/pSoup dual binary vector system.
  • expression vectors which place the inserted transgene(s) under the control of the constitutive CaMV 35S promoter and the RuBisCo promoter are employed.
  • a number of expression vectors which utilize the CaMV 35S and RuBsCo promoter are known and/or commercially available and/or derivable using ordinary skill in the art. Additionally, numerous promoters suitable for directing the expression of the transgene are known and may be used in the practice of the invention, as further described, infra.
  • the selected promoter(s) may be constitutive, non-specific promoters such as the Cauliflower Mosaic Virus 35S ribosomal promoter (CaMV 35S promoter), which is widely employed for the expression of transgenes in plants.
  • CaMV 35S promoter Cauliflower Mosaic Virus 35S ribosomal promoter
  • other strong constitutive promoters include without limitation the rice actin 1 promoter, the CaMV 19S promoter, the Ti plasmid nopaline synthase promoter, the alcohol dehydrogenase promoter and the sucrose synthase promoter.
  • a promoter based upon the desired plant cells to be transformed by the transgene construct, the desired expression level of the transgene, the desired tissue or subcellular compartment for transgene expression, the developmental stage targeted, and the like.
  • a leaf-specific promoter such as the ribulose bisphosphate carboxylase (RuBisCo) gene may be employed.
  • RuBisCo ribulose bisphosphate carboxylase
  • expressible nucleic acid constructs including GPT and GS1 transgenes under the control of a tomato RuBisCo promoter were prepared and used in the generation of transgenic plants or to assay for GPT activity in planta or in E. coli.
  • promoters of various seed storage protein genes may be employed.
  • a fruit-specific promoter such as tomato 2A1 1 may be used.
  • tissue specific promoters include the promoters encoding lectin (Vodkin et al., 1983, Cell 34:1023-31 ; Lindstrom et al., 1990, Developmental Genetics 1 1 :160-167), corn alcohol dehydrogenase 1 (Vogel et al, 1989, J. Cell. Biochem. (Suppl. 0) 13:Part D; Dennis et al., 1984, Nucl.
  • inducible promoter sequences may be employed in cases where it is desirable to regulate transgene expression as the transgenic plant regenerates, matures, flowers, etc.
  • inducible promoters include promoters of heat shock genes, protection responding genes (i.e., phenylalanine ammonia lyase; see, for example Bevan et al., 1989, EMBO J. 8(7): 899-906), wound responding genes (i.e., cell wall protein genes), chemically inducible genes (i.e., nitrate reductase, chitinase) and dark inducible genes (i.e., asparagine synthetase; see, for example U.S.
  • Patent No. 5,256,558 a number of plant nuclear genes are activated by light, including gene families encoding the major chlorophyll a/b binding proteins ⁇ cab) as well as the small subunit of ribulose-1 ,5-bisphosphate carboxylase ⁇ rbcS) (see, for example, Tobin and Silverthorne, 1985, Annu. Rev. Plant Physiol. 36: 569-593; Dean et al., 1989, Annu. Rev. Plant Physiol. 40: 415-439.).
  • inducible promoters include ABA- and turgor-inducible promoters, the auxin- binding protein gene promoter (Schwob et al., 1993, Plant J. 4(3): 423-432), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988, Genetics 1 19(1 ): 185-197); the MPI proteinase inhibitor promoter (Cordero et al., 1994, Plant J. 6(2): 141 -150), the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995, Plant Mol. Biol.
  • a 3' transcription termination sequence is incorporated downstream of the transgene in order to direct the termination of transcription and permit correct polyadenylation of the mRNA transcript.
  • Suitable transcription terminators are those which are known to function in plants, including without limitation, the nopaline synthase (NOS) and octopine synthase (OCS) genes of Agrobacterium tumefaciens, the T7 transcript from the octopine synthase gene, the 3' end of the protease inhibitor I or II genes from potato or tomato, the CaMV 35S terminator, the tml terminator and the pea rbcS E9 terminator.
  • a gene's native transcription terminator may be used. In specific embodiments, described by way of the Examples, infra, the nopaline synthase transcription terminator is employed.
  • Selectable markers are typically included in transgene expression vectors in order to provide a means for selecting transformants. While various types of markers are available, various negative selection markers are typically utilized, including those which confer resistance to a selection agent that inhibits or kills untransformed cells, such as genes which impart resistance to an antibiotic (such as kanamycin, gentamycin, anamycin, hygromycin and hygromycinB) or resistance to a herbicide (such as sulfonylurea, gulfosinate, phosphinothricin and glyphosate). Screenable markers include, for example, genes encoding ⁇ -glucuronidase (Jefferson, 1987, Plant Mol. Biol.
  • E. coli glucuronidase gene (gus, gusA or uidA) has become a widely used selection marker in plant transgenics, largely because of the glucuronidase enzyme's stability, high sensitivity and ease of detection (e.g., fluorometric, spectrophotometric, various histochemical methods). Moreover, there is essentially no detectable glucuronidase in most higher plant species.
  • transgene expression vector constructs of the invention into a plant or plant cell are well known to those skilled in the art, and any capable of transforming the target plant or plant cell may be utilized.
  • Agrobacterium- mediated transformation is perhaps the most common method utilized in plant transgenics, and protocols for Agrobacterium-med ⁇ ated transformation of a large number of plants are extensively described in the literature (see, for example, Agrobacterium Protocols, Wan, ed., Humana Press, 2 nd edition, 2006).
  • Agrobacterium tumefaciens is a Gram negative soil bacteria that causes tumors (Crown Gall disease) in a great many dicot species, via the insertion of a small segment of tumor-inducing DNA ( "T-DNA", 'transfer DNA') into the plant cell, which is incorporated at a semi-random location into the plant genome, and which eventually may become stably incorporated there.
  • T-DNA tumor-inducing DNA
  • Directly repeated DNA sequences, called T- DNA borders define the left and the right ends of the T-DNA.
  • the T-DNA can be physically separated from the remainder of the Ti-plasmid, creating a 'binary vector' system.
  • Agrobacterium transformation may be used for stably transforming dicots, monocots, and cells thereof (Rogers et al., 1986, Methods Enzymol., 1 18: 627-641 ; Hernalsteen et al., 1984, EMBO J., 3: 3039-3041 ; Hoykass-Van Slogteren et al., 1984, Nature, 31 1 : 763-764; Grimsley et al., 1987, Nature 325: 167-1679; Boulton et al., 1989, Plant Mol. Biol. 12: 31 -40; Gould et al., 1991 , Plant Physiol. 95: 426-434).
  • Agrobacterium- mediated transformation may be used to obtain transient expression of a transgene via the transcriptional competency of unincorporated transgene construct molecules (Helens et al., 2005, Plant Methods 1 :13).
  • Agrobacterium transformation vectors and methods have been described (Karimi et al., 2002, Trends Plant Sci. 7(5): 193-5), and many such vectors may be obtained commercially (for example, Invitrogen, Carlsbad, CA).
  • a growing number of "open-source" Agrobacterium transformation vectors are available (for example, pCambia vectors; Cambia, Canberra, Australia). See, also, subsection herein on TRANSGENE CONSTRUCTS, supra.
  • the Agrobacterium vector pTF101 .1 was employed.
  • This vector pTF101 .1 is a derivative of the pPZP binary vector (Hajdukiewicz et al 1994, Plant Mol. Biol. 25:989-994), which includes the right and left T-DNA border fragments from a nopaline strain of A. tumefaciens, a broad host origin of replication (pVS1 ) and a spectinomycin-resistant marker gene ⁇ aadA) for bacterial selection.
  • PEG polyethylene glycol
  • electroporation Paszkowski et al., 1984, EMBO J. 3: 2727-2722
  • Potrykus et al. 1985, Mol. Gen. Genet. 199: 169-177
  • Biolistic transformation involves injecting millions of DNA-coated metal particles into target cells or tissues using a biolistic device (or "gene gun"), several kinds of which are available commercially. Once inside the cell, the DNA elutes off the particles and a portion may be stably incorporated into one or more of the cell's chromosomes (for review, see Kikkert et al., 2005, Stable Transformation of Plant Cells by Particle Bombardment/Biolistics, in: Methods in Molecular Biology, vol. 286: Transgenic Plants: Methods and Protocols, Ed. L. Peha, Humana Press Inc., Totowa, NJ).
  • Electroporation is a technique that utilizes short, high-intensity electric fields to permeabilize reversibly the lipid bilayers of cell membranes (see, for example, Fisk and Dandekar, 2005, Introduction and Expression of Transgenes in Plant Protoplasts, in: Methods in Molecular Biology, vol. 286: Transgenic Plants: Methods and Protocols, Ed. L. Peha, Humana Press Inc., Totowa, NJ, pp. 79-90; Fromm et al.,1987, Electroporation of DNA and RNA into plant protoplasts, in Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press, London, UK, pp.
  • the technique operates by creating aqueous pores in the cell membrane, which are of sufficiently large size to allow DNA molecules (and other macromolecules) to enter the cell, where the transgene expression construct (as T- DNA) may be stably incorporated into plant genomic DNA, leading to the generation of transformed cells that can subsequently be regenerated into transgenic plants.
  • Newer transformation methods include so-called "floral dip” methods, which offer the promise of simplicity, without requiring plant tissue culture, as is the case with all other commonly used transformation methodologies (Bent et al., 2006, Arabidopsis thaliana Floral Dip Transformation Method, Methods Mol Biol, vol. 343: Agrobacterium Protocols, 2/e, volume 1 ; Ed., Wang, Humana Press Inc., Totowa, NJ, pp. 87-103; Clough and Bent, 1998, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16: 735- 743). However, with the exception of Arabidopsis, these methods have not been widely used across a broad spectrum of different plant species.
  • floral dip transformation is accomplished by dipping or spraying flowering plants in with an appropriate strain of Agrobacterium tumefaciens. Seeds collected from these T 0 plants are then germinated under selection to identify transgenic Ti individuals.
  • Example 16 demonstrated floral dip inoculation of Arabidopsis to generate transgenic Arabidopsis plants.
  • transformation methods include those in which the developing seeds or seedlings of plants are transformed using vectors such as Agrobacterium vectors.
  • vectors such as Agrobacterium vectors.
  • such vectors may be used to transform developing seeds by injecting a suspension or mixture of the vector (i.e., Agrobacteria) directly into the seed cavity of developing pods (i.e., pepper pods, bean pods, pea pods and the like).
  • Seedlings may be transformed as described for Alfalfa in Example 13.
  • Germinating seeds may be transformed as described for Camelina in Example 18.
  • Intra-fruit methods in which the vector is injected into fruit or developing fruit, may be used as described for Cantaloupe melons in Example 14 and pumpkins in Example 15.
  • Still other transformation methods include those in which the flower structure is targeted for vector inoculation, such as the flower inoculation methods described for beans in Examples 9 and 10, peas in Examples 1 1 and 12 and tomatoes in Example 17.
  • transgenes are most commonly inserted into the nuclear DNA of plant cells, transgenes may also be inserted into plastidic DNA (i.e., into the plastome of the chloroplast).
  • plastidic DNA i.e., into the plastome of the chloroplast.
  • Plastid transformation is more complex than cell nucleus transformation, due to the presence of many thousands of plastomes per cell (as high as 10,000).
  • Transplastomic lines are genetically stable only if all plastid copies are modified in the same way, i.e. uniformly. This is typically achieved through repeated regeneration under certain selection conditions to eliminate untransformed plastids, by segregating transplastomic and untransformed plastids, resulting in the selection of homoplasmic cells carrying the transgene construct and the selectable marker stably integrated therein. Plastid transformation has been successfully performed in various plant species, including tobacco, potatoes, oilseed rape, rice and Arabidopsis.
  • the transgene expression cassette is inserted into flanking sequences from the plastome. Using homologous recombination, the transgene expression cassette becomes integrated into the plastome via a natural recombination process.
  • the basic DNA delivery techniques for plastid transformation include particle bombardment of leaves or polyethylene glycol-mediated DNA transformation of protoplasts.
  • Transplastomic plants carrying transgenes in the plastome may be expressed at very high levels, due to the fact that many plastids (i.e., chloroplasts) per cell, each carrying many copies of the plastome.
  • the foregoing plant transformation methodologies may be used to introduce transgenes into a number of different plant cells and tissues, including without limitation, whole plants, tissue and organ explants including chloroplasts, flowering tissues and cells, protoplasts, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells, tissue cultured cells of any of the foregoing, any other cells from which a fertile regenerated transgenic plant may be generated.
  • Callus is initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation.
  • transformed plantlets derived from transformed cells or tissues are cultured in a root-permissive growth medium supplemented with the selective agent used in the transformation strategy (i.e., and antibiotic such as kanamycin). Once rooted, transformed plantlets are then transferred to soil and allowed to grow to maturity. Upon flowering, the mature plants are preferably selfed (self-fertilized), and the resultant seeds harvested and used to grow subsequent generations. Examples 3 - 6 describe the regeneration of transgenic tobacco and tomato plants.
  • T 0 transgenic plants may be used to generate subsequent generations (e.g., T ; T 2 , etc.) by selfing of primary or secondary transformants, or by sexual crossing of primary or secondary transformants with other plants (transformed or untransformed).
  • Example 7 For example, as described in Example 7, infra, individual plants over expressing the Alfalfa GS1 gene and outperforming wildtype plants were crossed with individual plants over-expressing the Arabidopsis GPT gene and outperforming wildtype plants, by simple sexual crossing using manual pollen transfer. Reciprocal crosses were made such that each plant served as the male in a set of crosses and each plant served as the female in a second set of crosses. During the mature plant growth stage, the plants are typically examined for growth phenotype, CO 2 fixation rate, etc. (see following subsection).
  • Transgenic plants may be selected, screened and characterized using standard methodologies.
  • the preferred transgenic plants of the invention will exhibit one or more phenotypic characteristics indicative of enhanced growth and/or other desirable agronomic properties.
  • Transgenic plants are typically regenerated under selective pressure in order to select transformants prior to creating subsequent transgenic plant generations.
  • the selective pressure used may be employed beyond To generations in order to ensure the presence of the desired transgene expression construct or cassette.
  • T 0 transformed plant cells, calli, tissues or plants may be identified and isolated by selecting or screening for the genetic composition of and/or the phenotypic characteristics encoded by marker genes contained in the transgene expression construct used for the transformation. For example, selection may be conducted by growing potentially-transformed plants, tissues or cells in a growth medium containing a repressive amount of antibiotic or herbicide to which the transforming genetic construct can impart resistance. Further, the transformed plant cells, tissues and plants can be identified by screening for the activity of marker genes (i.e., ⁇ - glucuronidase) which may be present in the transgene expression construct. Various physical and biochemical methods may be employed for identifying plants containing the desired transgene expression construct, as is well known.
  • marker genes i.e., ⁇ - glucuronidase
  • Examples of such methods include Southern blot analysis or various nucleic acid amplification methods (i.e., PCR) for identifying the transgene, transgene expression construct or elements thereof, Northern blotting, S1 RNase protection, reverse transcriptase PCR (RT-PCR) amplification for detecting and determining the RNA transcription products, and protein gel electrophoresis, Western blotting, immunoprecipitation, enzyme immunoassay, and the like may be used for identifying the protein encoded and expressed by the transgene.
  • PCR nucleic acid amplification methods
  • RT-PCR reverse transcriptase PCR
  • expression levels of genes, proteins and/or metabolic compounds that are know to be modulated by transgene expression in the target plant may be used to identify transformants.
  • increased levels of the signal metabolite 2-oxoglutaramate may be used to screen for desirable transformants, as exemplified in the Examples.
  • increased levels of GPT and/or GS activity may be assayed, as exemplified in the Examples.
  • the transformed plants of the invention may be screened for enhanced growth and/or other desirable agronomic characteristics. Indeed, some degree of phenotypic screening is generally desirable in order to identify transformed lines with the fastest relative growth rates, the highest seed yields, etc., particularly when identifying plants for subsequent selfing, cross-breeding and back-crossing.
  • Various parameters may be used for this purpose, including without limitation, growth rates, total fresh weights, dry weights, seed and fruit yields (number, weight), seed and/or seed pod sizes, seed pod yields (e.g., number, weight), leaf sizes, plant sizes, increased flowering, time to flowering, overall protein content (in seeds, fruits, plant tissues), specific protein content (i.e., GS), nitrogen content, free amino acid, and specific metabolic compound levels (i.e., 2-oxoglutaramate).
  • growth rates total fresh weights, dry weights, seed and fruit yields (number, weight), seed and/or seed pod sizes, seed pod yields (e.g., number, weight), leaf sizes, plant sizes, increased flowering, time to flowering, overall protein content (in seeds, fruits, plant tissues), specific protein content (i.e., GS), nitrogen content, free amino acid, and specific metabolic compound levels (i.e., 2-oxoglutaramate).
  • these phenotypic measurements are compared with those obtained from
  • the measurement of the chosen phenotypic characteristic(s) in the target transgenic plant is done in parallel with measurement of the same characteristic(s) in a normal or parental plant.
  • multiple plants are used to establish the phenotypic desirability and/or superiority of the transgenic plant in respect of any particular phenotypic characteristic.
  • initial transformants are selected and then used to generate Ti and subsequent generations by selfing (self-fertilization), until the transgene genotype breeds true (i.e., the plant is homozygous for the transgene).
  • self-fertilization i.e., the plant is homozygous for the transgene.
  • this is accomplished by screening at each generation for the desired traits and selfing those individuals, often repeatedly (i.e., 3 or 4 generations).
  • transgenic plant lines propagated through at least one sexual generation See tobacco, Arabidopsis, Tomato
  • Stable transgenic lines may be crossed and back-crossed to create varieties with any number of desired traits, including those with stacked transgenes, multiple copies of a transgene, etc.
  • Various common breeding methods are well known to those skilled in the art (see, e.g., Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1 987)).
  • stable transgenic plants may be further modified genetically, by transforming such plants with further transgenes or additional copies of the parental transgene.
  • the invention provides transgenic plants characterized by increased nitrogen use efficiency.
  • Nitrogen use efficiency may be expressed as plant yield per given amount of nitrogen.
  • the transgene and control plants all received the same nutrient solutions in the same amounts.
  • the transgenic plants were consistently characterized by higher yields, and thus have higher nitrogen use efficiencies.
  • the invention provides transgenic plants and seeds thereof with increased tolerance to high salt growth conditions.
  • This aspect of the invention is exemplified by Example 24, which describes the germination of transgenic tobacco plant seeds in very high salt conditions (200 mM NaCI). While counterpart wild type tobacco seeds germinated at a rate of only about 1 0%, on average, the transgenic tobacco seeds achieved nearly the same rate of germination obtained under no salt conditions for both transgenic and wild type seeds, or about 92%.
  • the invention provides novel transgenic plants exhibiting substantially enhanced agronomic characteristics, including faster growth, faster relative growth rate, greater mature plant fresh weight and total biomass, earlier and more abundant flowering, and greater fruit, pod and seed yields.
  • the transgenic plants of the invention are generated by introducing into a plant one or more expressible genetic constructs capable of driving the expression of one or more polynucleotides encoding either a glutamine phenylpyruvate transaminase (GPT) or both a glutamine synthetase (GS) and glutamine phenylpyruvate transaminase (GPT).
  • the invention is exemplified, for example, by the generation of transgenic tobacco plants carrying and expressing the heterologous Arabidopsis GPT gene (Example 2, infra).
  • single-transgene parental lines carrying either a GPT or GS1 transgene coding sequence are generated, preferably selfed until homozygous for the transgene, then crossed to generate progeny plants containing both transgenes. It is expected that all plant species also contain a GPT homolog which functions in the same metabolic pathway, namely the biosynthesis of the signal metabolite 2-hydroxy- 5-oxoproline.
  • any plant gene encoding a GPT homolog or functional variants thereof may be useful in the generation of transgenic plants of this invention.
  • the transgenic plants of the invention may be any vascular plant of the phylum Tracheophyta, including angiosperms and gymnosperms.
  • Angiosperms may be a monocotyledonous (monocot) or a dicotyledonous (dicot) plant.
  • Important monocots include those of the grass families, such as the family Poaceae and Gramineae, including plants of the genus Avena ⁇ Avena sativa, oats), genus Hordeum (i.e., Hordeum vulgare, Barley), genus Oryza (i.e., Oryza sativa, rice, cultivated rice varieties), genus Panicum ⁇ Panicum spp., Panicum virgatum, Switchgrass), genus Phleum ⁇ Phleum pratense, Timothy-grass), genus Saccharum (i.e., Saccharum officinarum, Saccharum spontaneum, hybrids thereof, Sugarcane), genus Secale (i.e., Secale cereale, Rye), genus Sorghum ⁇ Sorghum vulgare, Sorghum), genus Triticum (wheat, various classes, including T.
  • genus Hordeum i.e., Hordeum
  • genus Fagopyrum buckwheat, including F. esculentum
  • genus Triticosecale Triticale, various hybrids of wheat and rye
  • genus Chenopodium quinoa, including C. quinoa
  • genus Zea i.e., Zea mays, numerous varieties
  • millets i.e., Pennisetum glaucum
  • Digitaria Digitaria
  • Important dicots include those of the family Solanaceae, such as plants of the genus Lycopersicon ⁇ Lycopersicon esculentum, tomato), genus Capiscum ⁇ Capsicum annuum, peppers), genus Solanum ⁇ Solanum tuberosum, potato, S. lycopersicum, tomato); genus Manihot (cassava, M. esculenta), genus Ipomoea (sweet potato, /. batatas), genus Olea (olives, including O. europaea); plants of the Gossypium family (i.e., Gossypium spp., G. hirsutum, G.
  • Solanaceae such as plants of the genus Lycopersicon ⁇ Lycopersicon esculentum, tomato), genus Capiscum ⁇ Capsicum annuum, peppers), genus Solanum ⁇ Solanum tuberos
  • Legumes family Fabaceae
  • peas ⁇ Pisum spp, P. sativum beans ⁇ Glycine spp., Glycine max(soybean); Phaseolus vulgaris, common beans, Vigna radiata, mung bean), chickpeas ⁇ Cicer arietinum)), lentils ⁇ Lens culinaris), peanuts ⁇ Arachis hypogaea
  • genus Wf/ ' s (grape, including Vitis vinifera), and plants of the genus Brassica (family Brassicaceae, i.e., broccoli, brussel sprouts, cabbage, swede, turnip, rapeseed B. napus, and cauliflower).
  • family Brassicaceae i.e., broccoli, brussel sprouts, cabbage, swede, turnip, rapeseed B. napus, and cauliflower.
  • Other specific plants which may be transformed to generate the transgenic plants of the invention include various other fruits and vegetables, such as apples, asparagus, avocado, banana, blackberry, blueberry, brussel sprout, cabbage, cotton, canola, carrots, radish, cucumbers, cherries, cranberries, cantaloupes, eggplant, grapefruit, lemons, limes, nectarines, oranges, peaches, pineapples, pears, plums, tangelos, tangerines, papaya, mango, strawberry, raspberry, lettuce, onion, grape, kiwi fruit, okra, parsnips, pumpkins, and spinach.
  • fruits and vegetables such as apples, asparagus, avocado, banana, blackberry, blueberry, brussel sprout, cabbage, cotton, canola, carrots, radish, cucumbers, cherries, cranberries, cantaloupes, eggplant, grapefruit, lemons, limes, nectarines, oranges, peaches, pineapples, pears, plums, tangelos, tangerines, papaya,
  • various flowering plants, trees and ornamental plants may be used to generate transgenic varietals, including without limitation lily, carnation, chrysanthemum, petunia, geranium, violet, gladioli, lupine, orchid and lilac.
  • one or more copies of the expressible genetic construct become integrated into the host plant genome, thereby providing increased GPT or GS and GPT enzyme capacity into the plant, which serves to mediate increased synthesis of 2-oxoglutaramate, which in turn signals metabolic gene expression, resulting in increased plant growth and the enhancement other agronomic characteristics.
  • 2-oxoglutaramate is a metabolite which is an extremely potent effector of gene expression, metabolism and plant growth (U.S. Patent No.
  • a nucleic acid molecule encoding the Arabidopsis glutamine phenylpyruvate transaminase (GPT) enzyme (see Example 1 , infra), and have demonstrated for the first time that the expressed recombinant enzyme is active and capable of catalyzing the synthesis of the signal metabolite, 2-oxoglutaramate (Example 2, infra). Further, applicants have demonstrated for the first time that over-expression of the Arabidopsis glutamine transaminase gene in a transformed heterologous plant results in enhanced CO 2 fixation rates and increased growth characteristics (Example 3, infra).
  • GPT Arabidopsis glutamine phenylpyruvate transaminase
  • transgenic tobacco plants As disclosed herein (see Example 3, infra), over-expression of a transgene including the full-length Arabidopsis GPT coding sequence in transgenic tobacco plants also results in faster CO 2 fixation, and increased levels of total protein, glutamine and 2- oxoglutaramate. These transgenic plants also grow faster than wild-type plants (FIG. 4).
  • tomato plants transformed with the Arabidopsis GPT transgene showed significant enhancement of growth rate, flowering, and seed yield in relation to wild type control plants (FIG. 5 and Example 4, infra).
  • a first set of parental single-transgene tobacco plant lines carrying the Alfalfa GS1 gene, including 5' and 3' untranslated regions, were generated using Agrobacterium mediated gene transformation, under selective pressure, together with screening for the fastest growing phenotype, and selfing to transgene/phenotype homozygosity (see Example 5, infra).
  • a second set of parental single-transgene tobacco plant lines carrying the full length coding sequence of Arabidopsis GPT were generated in the same manner (Example 3, infra). High growth rate performing plants from each of the parental lines were then sexually crossed to yield progeny lines (Example 7, infra).
  • FIG. 7 shows photographs of double-transgene progeny from single-transgene GS1 X GPT plant crosses, relative to wild type and single-transgene parental plants.
  • FIG. 8 shows photographs comparing leaf sizes of double-transgene progeny and wild type plants.
  • Experimentally observed growth rates in these double transgenic plants ranged between 200% and 300% over wild-type plants (Example 7, infra).
  • total biomass levels increased substantially in the double-transgene plants, with whole plant fresh weights typically being about two to three times the wild-type plant weights.
  • seed yields showed similar increases in the double-transgene plants, with seed pod production typically two to three times the wild type average, and overall seed yields exceeding wild-type plant yields by 300-400%.
  • transgenic tobacco plants In addition to the transgenic tobacco plants referenced above, various other species of transgenic plants including GPT and showing enhanced growth characteristics have been generated in two species of Tomato, Pepper, Beans, Cowpea, Alfalfa, Cantaloupe, Pumpkin, Arabidopsis and Camilena (see co-pending US Application Number 12/551 ,271 , filed August 31 , 2009, the contents of which are incorporated herein by reference in its entirety). Furthermore, various other species of transgenic plants including GPT and GS transgenes are specifically exemplified herein.
  • transgenic plants showing enhanced growth characteristics have been generated in two species of Tomato (see Examples 4 and 17), Pepper (Example 8), Beans (Examples 9 and 10), Cowpea (Examples 1 1 and 12), Alfalfa (Example 13), Cantaloupe (Example 14), Pumpkin (Example 15), Arabidopsis (Example 16) and Camilena (Example 18).
  • These transgenic plants of the invention were generated using a variety of transformation methodologies, including Agrobacterium-mediated callus, floral dip, seed inoculation, pod inoculation, and direct flower inoculation, as well as combinations thereof, and via sexual crosses of single transgene plants, as exemplified herein.
  • Different GPT or GPT and GS transgenes were successfully employed in generating the transgenic plants of the invention, as exemplified herein.
  • GPT GPT
  • GPT and GS polynucleotides may be used to generate the transgenic plants of the invention. Both GS1 and GPT proteins are highly conserved among various plant species, and it is evident from the experimental data disclosed herein that closely-related non-plant GPTs may be used as well (e.g., Danio rerio GPT). With respect to GPT, numerous GPT polynucleotides derived from different species have been shown to be active and useful as GPT transgenes. Similarly, different GS polynucleotides may be used, including without limitation any plant GS1 encoding polynucleotide that generates GS activity in a host cell transformed with an expressible GS1 construct.
  • the GPT transgene is a GPT polynucleotide encoding an Arabidopsis derived GPT, such as the GPT of SEQ ID NO: 2, SEQ ID NO: 21 and SEQ ID NO: 30.
  • the GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 1 ; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 1 , and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity; and a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 truncated at its amino terminus by between 30 to 56 amino acid residues, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.
  • the Arabidopsis derived GPT is combined with a GS transgene, whereby the GS transgene is a GS polynucleotide encoding an Alfalfa derived GS1 (i.e., SEQ ID NO: 4) or an Arabidopsis derived GS1 (SEQ ID NO: 7).
  • the GS transgene is a GS polynucleotide encoding an Alfalfa derived GS1 (i.e., SEQ ID NO: 4) or an Arabidopsis derived GS1 (SEQ ID NO: 7).
  • the GS1 transgene may be encoded by the polynucleotide of SEQ ID NO: 3 or SEQ ID NO: 6 or a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 3 or SEQ ID NO: 6, and encoding a polypeptide having GS activity; and a nucleotide sequence encoding the polypeptide of SEQ ID NO: 4 or 7, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GS activity.
  • the GPT transgene is a GPT polynucleotide encoding a Grape derived GPT, such as the Grape GPTs of SEQ ID NO: 9 and SEQ ID NO: 31 .
  • the GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 8; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 8, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 9 or SEQ ID NO: 31 , or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.
  • the Grape derived GPT is combined with a GS transgene, whereby the GS transgene is a GS1 polynucleotide.
  • the GPT transgene is a GPT polynucleotide encoding a Rice derived GPT, such as the Rice GPTs of SEQ ID NO: 1 1 and SEQ ID NO: 32.
  • the GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 10; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 10, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 1 1 or SEQ ID NO: 32, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.
  • the Rice derived GPT is combined with a GS transgene, whereby the GS transgene is a GS1 polynucleotide.
  • the GPT transgene is a GPT polynucleotide encoding a Soybean derived GPT, such as the Soybean GPTs of SEQ ID NO: 13, SEQ ID NO: 33 or SEQ ID NO: 33 with a further Isoleucine at the N-terminus of the sequence.
  • the GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 12; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 12, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 13 or SEQ ID NO: 33 or SEQ ID NO: 33 with a further Isoleucine at the N-terminus of the sequence, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.
  • the Soybean derived GPT is combined with a GS transgene, whereby the GS transgene is a GS1 polynucleotide.
  • the GPT transgene is a GPT polynucleotide encoding a Barley derived GPT, such as the Barley GPTs of SEQ ID NO: 15 and SEQ ID NO: 34.
  • the GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 14; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 10, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 15 or SEQ ID NO: 34, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.
  • the Barley derived GPT is combined with a GS transgene, whereby the GS transgene is a GS1 polynucleotide.
  • the GPT transgene is a GPT polynucleotide encoding a Zebra fish derived GPT, such as the Zebra fish GPTs of SEQ ID NO: 17 and SEQ ID NO: 35.
  • the GPT transgene may be encoded by the nucleotide sequence of SEQ ID NO: 16; a nucleotide sequence having at least 75% and more preferably at least 80% identity to SEQ ID NO: 16, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding the polypeptide of SEQ ID NO: 17 or SEQ ID NO: 35, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.
  • the Zebra fish derived GPT is combined with a GS transgene, whereby the GS transgene is a GS1 polynucleotide.
  • the GPT transgene is a GPT polynucleotide encoding a Bamboo derived GPT, such as the Bamboo GPT of SEQ ID NO: 36.
  • the GPT transgene may be encoded by a nucleotide sequence encoding the polypeptide of SEQ ID NO: 36, or a polypeptide having at least 75% and more preferably at least 80% sequence identity thereto which has GPT activity.
  • the bamboo derived GPT is combined with a GS transgene, whereby the GS transgene is a GS1 polynucleotide.
  • GPT polynucleotides suitable for use as GPT transgenes in the practice of the invention may be obtained by various means, as will be appreciated by one skilled in the art, tested for the ability to direct the expression of a GPT with GPT activity in a recombinant expression system (i.e., E. coli (see Examples 20-23), in a transient in planta expression system (see Example 19), or in a transgenic plant (see Examples 1 -18).
  • a recombinant expression system i.e., E. coli (see Examples 20-23)
  • transient in planta expression system see Example 19
  • transgenic plant see Examples 1 -18.
  • the disclosure herein provides materials derived from the transgenic plants or parts of the plants described herein.
  • the invention provides food products including the transgenic soybean or rice plants of the invention in whole or in parts, or including ingredients derived from transgenic soybean or rice plants of the invention.
  • Specific embodiments of the ingredients derived from transgenic soybean or rice plants of the invention include meal, oil, milk, flour, tofu, curd, ice cream, cereal, vinegar, or wine.
  • EXAMPLE 1 ISOLATION OF ARABIDOPSIS GLUAMINE PHENYLPYRUVATE TRANSAMINASE (GPT) GENE:
  • cysteine conjugate ⁇ -lyase also referred in the literature as cysteine conjugate ⁇ -lyase, kyneurenine aminotransferase, glutamine phenylpyruvate transaminase, and other names
  • cysteine conjugate ⁇ -lyase had been shown to be involved in processing of cysteine conjugates of halogenated xenobiotics (Perry et al., 1995, FEBS Letters 360:277-280).
  • human cysteine conjugate ⁇ -lyase has a detoxifying activity in humans, and in animals (Cooper and Meister, 1977, supra). Nevertheless, the potential involvement of this protein in the synthesis of 2-oxoglutaramate was of interest.
  • primers were designed to incorporate Cla I (ATCGAT) and Kpn I (GGTACC) restriction sites to facilitate subsequent subcloning into expression vectors for generating transgenic plants.
  • Takara ExTaq DNA polymerase enzyme was used for high fidelity PCR using the following conditions: initial denaturing 94°C for 4 minutes, 30 cycles of 94°C 30 second, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, with a final extension of 72°C for 7 minutes.
  • the amplification product was digested with Cla I and Kpn I restriction enzymes, isolated from an agarose gel electrophoresis and ligated into vector pMon316 (Rogers, et. al.
  • a 1 .3 kb cDNA was isolated and sequenced, and found to encode a full length protein of 440 amino acids in length, including a putative chloroplast signal sequence.
  • ARABIDOPSIS GLUTAMINE PHENYLPYRUVATE TRANSAMINASE GPT: To test whether the protein encoded by the cDNA isolated as described in Example 1 , supra, is capable of catalyzing the synthesis of 2-oxoglutaramate, the cDNA was expressed in E. coli, purified, and assayed for its ability to synthesize 2- oxoglutaramate using a standard method.
  • the resulting purified protein was added to a reaction mixture containing 150 mM Tris-HCI, pH 8.5, 1 mM beta mercaptoethanol, 200 mM glutamine, 100 mM glyoxylate and 200 ⁇ pyridoxal 5'-phosphate.
  • the reaction mixture without added test protein was used as a control.
  • Test and control reaction mixtures were incubated at 37°C for 20 hours, and then clarified by centrifugation to remove precipitated material. Supernatants were tested for the presence and amount of 2- oxoglutaramate using 13 C NMR with authentic chemically synthesized 2- oxoglutaramate as a reference.
  • the products of the reaction are 2-oxoglutaramate and glycine, while the substrates (glutamine and glyoxylate) diminish in abundance.
  • the cyclic 2-oxoglutaramate gives rise to a distinctive signal allowing it to be readily distinguished from the open chain glutamine precursor.
  • An alternative assay for GPT activity uses HPLC to determine 2-oxoglutaramate production, following a modification of Calderon et al., 1985, J Bacteriol 161 (2): 807- 809. Briefly, a modified extraction buffer consisting of 25 mM Tris-HCI pH 8.5, 1 mM EDTA, 20 ⁇ FAD, 10 mM Cysteine, and -1 .5% (v/v) Mercaptoethanol. Tissue samples from the test material (i.e., plant tissue) are added to the extraction buffer at approximately a 1 /3 ratio (w/v), incubated for 30 minutes at 37°C, and stopped with 200 ⁇ of 20% TCA.
  • the assay mixture is centrifuged and the supernatant used to quantify 2-oxoglutaramate by HPLC, using an ION-300 7.8mm ID X 30 cm L column, with a mobile phase in 0.01 N H2SO4, a flow rate of approximately 0.2 ml/min, at 40°C. Injection volume is approximately 20 ⁇ , and retention time between about 38 and 39 minutes. Detection is achieved with 210nm UV light. Results Using NMR Assay:
  • test protein was able to catalyze the synthesis of 2- oxoglutaramate. Therefore, these data indicate that the isolated cDNA encodes a glutamine phenylpyruvate transaminase that is directly involved in the synthesis of 2- oxoglutaramate in plants. Accordingly, the test protein was designated Arabidopsis glutamine phenylpyruvate transaminase, or "GPT".
  • the nucleotide sequence of the Arabidopsis GPT coding sequence is shown in the Table of Sequences, SEQ ID NO. 1 .
  • the translated amino acid sequence of the GPT protein is shown in SEQ ID NO. 2.
  • EXAMPLE 3 CREATION OF TRANSGENIC TOBACCO PLANTS OVER- EXPRESSING ARABIDOPSIS GPT:
  • the plant expression vector pMon316-PJU was constructed as follows.
  • the isolated cDNA encoding Arabidopsis GPT (Example 1 ) was cloned into the Clal-Kpnl polylinker site of the pMON316 vector, which places the GPT gene under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) transcriptional terminator.
  • CaMV constitutive cauliflower mosaic virus
  • NOS nopaline synthase
  • a kanamycin resistance gene was included to provide a selectable marker.
  • pMON-PJU and a control vector pMon316 were transferred to Agrobacterium tumefaciens strain pTiTT37ASE using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159), followed by plating on LB plates containing the antibiotics spectinomycin (100 micro gm / ml) and kanamycin (50 micro gm / ml). Antibiotic resistant colonies of Agrobacterium were examined by PCR to assure that they contained plasmid. Nicotiana tabacum cv. Xanthi plants were transformed with pMON-PJU transformed Agrobacteria using the leaf disc transformation system of Horsch et. al.
  • Seeds harvested form the T 0 generation of the transgenic tobacco plants were germinated on M&S media containing kanamycin (100 mg / L) to enrich for the transgene. At least one fourth of the seeds did not germinate on this media (kanamycin is expected to inhibit germination of the seeds without resistance that would have been produced as a result of normal genetic segregation of the gene) and more than half of the remaining seeds were removed because of demonstrated sensitivity (even mild) to the kanamycin.
  • Ti generation The surviving plants (Ti generation) were thriving and these plants were then selfed to produce seeds for the T 2 generation. Seeds from the Ti generation were germinated on MS media supplemented for the transformant lines with kanamycin (10mg/liter). After 14 days they were transferred to sand and provided quarter strength Hoagland's nutrient solution supplemented with 25 mM potassium nitrate. They were allowed to grow at 24°C with a photoperiod of 16 h light and 8 hr dark with a light intensity of 900 micromoles per meter squared per second. They were harvested 14 days after being transferred to the sand culture. Characterization of GPT Transgenic Plants:
  • FIG. 4 a photograph of the GPT transgenic plant compared to a wild type control plant is shown in FIG. 4 (together with GS1 transgenic tobacco plant, see Example 5).
  • the GPT transgenic tobacco plants showed enhanced growth characteristics.
  • the GPT transgenic plants exhibited a greater than 50% increase in the rate of CO 2 fixation, and a greater than two-fold increase in glutamine synthetase activity in leaf tissue, relative to wild type control plants.
  • the leaf-to-root GS ratio increased by almost three-fold in the transaminase transgenic plants relative to wild type control.
  • PN1 lines were produced by regeneration after transformation using a construct without inserted gene. A control against the processes of regeneration and transformation.
  • PN 9 lines were produced by regeneration after transformation using a construct with the Arabidopsis GPT gene.
  • Transgenic Lycopersicon esculentum (Micro-Tom Tomato) plants carrying the Arabidopsis GPT transgene were generated using the vectors and methods described in Example 3. T 0 transgenic tomato plants were generated and grown to maturity. Initial growth characteristic data of the GPT transgenic tomato plants is presented in Table II. The transgenic plants showed significant enhancement of growth rate, flowering, and seed yield in relation to wild type control plants. In addition, the transgenic plants developed multiple main stems, whereas wild type plants developed with a single main stem. A photograph of a GPT transgenic tomato plant compared to a wild type plant is presented in FIG. 5 (together with GS1 transgenic tomato plants, see Example 6).
  • EXAMPLE 5 GENERATION OF TRANSGENIC TOBACCO PLANTS OVEREXPRESSING ALFALFA GS1 :
  • Transgenic tobacco plants overexpressing the Alfalfa GS1 gene were generated as previously described (Temple et al., 1993, Mol. Gen. Genetics 236: 315-325). Briefly, the plant expression vector pGS1 1 1 was constructed by inserting the entire coding sequence together with extensive regions of both the 5' and 3' untranslated regions of the Alfalfa GS1 gene [SEQ ID NO: 3] (DasSarma at al., 1986, Science, Vol 232, Issue 4755, 1242-1244) into pMON316 (Rogers et al., 1987, supra), placing the transgene under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) transcriptional terminator. A kanamycin resistance gene was included to provide a selectable marker.
  • CaMV constitutive cauliflower mosaic virus
  • NOS nopaline synthase
  • pGS1 1 1 was transferred to Agrobacterium tumefaciens strain pTiTT37ASE using triparental mating as described (Rogers et al., 1987, supra; Unkefer et al., U.S. Patent No. 6,555,500). Nicotiana tabacum cv. Xanthi plants were transformed with pGS1 1 1 transformed Agrobacteria using the leaf disc transformation system of Horsch et. al. (Horsch et al.,1995, Science 227:1229-1231 ). Transformants were selected and regenerated on MS medium containing 100 ⁇ g/ml kanamycin.
  • Transgenic Lycopersicon esculentum (Micro-Tom Tomato) plants carrying the Alfalfa GS1 transgene were generated using the vector described in Example 5 and a transformation protocol essentially as described (Sun et al., 2006. Plant Cell Physiol. 46(3) 426-31 ). T 0 transgenic tomato plants were generated and grown to maturity. Initial growth characteristic data of the GPT transgenic tomato plants is presented in Table III. The transgenic plants showed significant enhancement of growth rate, flowering, and seed yield in relation to wild type control plants. In addition, the transgenic plants developed multiple main stems, whereas wild type plants developed with a single main stem. A photograph of a GS1 transgenic tomato plant compared to a wild type plant is presented in FIG. 5 (together with GPT transgenic tomato plant, see Example 4).
  • EXAMPLE 7 GENERATION OF DOUBLE TRANSGENIC TOBACCO PLANTS CARRYING GS1 AND GPT TRANSGENES:
  • GS activity was extracted from fresh plant tissue after grinding in cold 50 mM Imidazole, pH 7.5 containing 10 mM MgCI2, and 12.5 mM mercaptoethanol in a ratio of 3 ml per gram of tissue.
  • the extract was clarified by centrifugation and used in the assay.
  • GPT activity was assayed as described in Calderon and Mora, 1985, Journal Bacteriology 161 :807-809. GS activity was measured as described in Shapiro and Stadtmann, 1970, Methods in Enzymology 17A: 910-922. Both assays involve an incubation with substrates and cofactor at the proper pH. Detection was by HPLC.
  • Results The results are presented in two ways. First, specific growth characteristics are tabulated in Tables IV.A and IV. B (biomass, seed yields, growth rate, GS activity, GPT activity, 2-oxoglutaramate activity, etc). Second, photographs of progeny plants and their leaves are shown in comparison to single-transgene and wild type plants and leaves are presented in FIG. 7 and FIG. 8, which show much larger whole plants, larger leaves, and earlier and/or more abundant flowering in comparison to the parental single-transgene plants and wild type control plants.
  • double-transgene progeny plants form these crosses showed tremendous increases total biomass (fresh weight), with fresh weights ranging from 45-89 grams per individual progeny plant, compared to a range of only 19-24 grams per individual wild type plant, representing on average, about a two- to three-fold increase over wild type plants, and representing at the high end, an astonishing fourfold increase in biomass over wild type plants.
  • fresh weight ranging from 45-89 grams per individual progeny plant, compared to a range of only 19-24 grams per individual wild type plant, representing on average, about a two- to three-fold increase over wild type plants, and representing at the high end, an astonishing fourfold increase in biomass over wild type plants.
  • the average individual plant biomass was about 2.75 times that of the average wild type control plant.
  • Four of the progeny lines showed approximately 2.5 fold greater average per plant fresh weights, while two lines showed over three-fold greater fresh weights in comparison to wild type plants.
  • the double-transgene progeny plants In comparison to the single-transgene parental lines, the double-transgene progeny plants also showed far more than an additive growth enhancement. Whereas GPT single-transgene lines show as much as about a 50% increase over wild type biomass, and GS1 single-transgene lines as much as a 66% increase, progeny plants averaged almost a 200% increase over wild type plants.
  • the double transgene progeny plants flowered earlier and more prolifically than either the wild type or single transgene parental lines, and produced a far greater number of seed pods as well as total number of seeds per plant.
  • Table IV.A on average, the double-transgene progeny produced over twice the number of seed pods produced by wild type plants, with two of the high producer plants generating over three times the number of seed pods compared to wild type.
  • Total seed yield in progeny plants measured on a per plant weight basis, ranged from about double to nearly quadruple the number produced in wild type plants.
  • Table IV. B shows growth rate, biomass and yield, and biochemical characteristics of Line XX (Line 3 further selfed) compared to the single transgene line expressing GS1 and wild type control tobacco. All parameters are greatly increased in the double transgenic plant (Line XX). Notably, 2-oxoglutaramate activity was almost 17-fold higher, and seed yield and foliar biomass was three-fold higher, in Line XX plants versus control plants.
  • EXAMPLE 8 GENERATION OF DOUBLE TRANSGENIC PEPPER PLANTS CARRYING GS1 AND GPT TRANSGENES:
  • Big Jim chili pepper plants (New Mexico varietal) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter, and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter, using Agrobacterium-mediated transfer to seed pods. After 3 days, seeds were harvested and used to generate TO plants and screened for transformants. The resulting double-transgenic plants showed higher pod yields, faster growth rates, and greater biomass yields in comparison to the control plants.
  • Solanaceae Capisicum Pepper plants (“Big Jim” varietal) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON (see Example 3), and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (Tomato rubisco rbcS3C promoter: Kyozulka et al., 1993, Plant Physiol. 103: 991 -1000; SEQ ID NO: 22; vector construct of SEQ ID NO: 6), using Agrobacterium-mediated transfer to seed pods.
  • the Cambia 1201 or 1305.1 vectors were constructed according to standard cloning methods (Sambrook et al., 1989, supra, Saiki et al., 1988, Science 239: 487-491 ).
  • the vector is supplied with a 35S CaMV promoter; that promoter was replaced with RcbS-3C promoter from tomato to control the expression of the target gene.
  • the Cambia 1201 vectors contain bacterial chlorophenicol and plant hygromycin resistance selectable marker genes.
  • the Cambia 1305.1 vectors contain bacterial chlorophenicol and hygromycin resistance selectable marker genes.
  • transgene expression vectors pMON (GPT transgene) and pCambia 1201 (GS transgene) were transferred to separate Agrobacterium tumefaciens strain LBA4404 cultures using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159).
  • Transformed Agrobacterium were selected on media containing 50 ⁇ g/ml of either streptamycin for pMON constructs or chloroamphenicol for the Cambia constructs.
  • Transformed Agrobacterium cells were grown in LB culture media containing 25 ⁇ g/ml of antibiotic for 36 hours. At the end of the 36 hr growth period cells were collected by centrifugation and cells from each transformation were resuspended in 100 ml LB broth without antibiotic.
  • Pepper plants were then transformed with a mixture of the resulting Agrobacterium cell suspensions using a transformation protocol in which the Agrobacterium is injected directly into the seed cavity of developing pods. Briefly, developing pods were injected with the 200 ml mixture in order to inoculate immature seeds with the Agrobacteria essentially as described (Wang and Waterhouse, 1997, Plant Mol. Biol. Reporter 15: 209-215). In order to induce Agrobacteria virulence and improve transformation efficiencies, 10 ⁇ g/ml acetosyringonone was added to the Agrobacteria cultures prior to pod inoculations (see, Sheikholeslam and Weeks, 1986, Plant Mol. Biol. 8: 291 -298).
  • pods were injected with a liberal quantity of the Agrobacterium vector mixture, and left to incubate for about 3 days. Seeds were then harvested and germinated, and developing plants observed for phenotypic characteristics including growth and antibiotic resistance. Plants carrying the transgenes were green, whereas untransformed plants showed signs of chlorosis in leaf tips. Vigorous growing transformants were further cultivated and compared to wild type pepper plants grown under identical conditions.
  • FIG. 9 shows a photograph of a GPT+GS double transgenic pepper plant compared to a control plant grown for the same time under identical conditions. This photograph shows tremendous pepper yield in the transgenic line compared to the control plant.
  • Table V presents biomass yield and GS activity, as well as transgene genotyping, in the transgenic lines compared to the wild type control. Referring to Table V, double- transgene progeny plants showed tremendous increases total biomass (fresh weight), with fresh weights, ranging from 393 - 662 grams per individual transgenic plant, compared to an average of 328 grams per wild type plant. Transgenic line A5 produced more than twice the total biomass of the controls. Moreover, pepper yields in the transgenic lines were greatly improved over wild type plants, and were 50% greater than control plants (on average). Notably, one of the transgene lines produced twice as many peppers as the control plant average.
  • yellow wax bean plants ⁇ Phaseolus vulgaris were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pCambia 1 201 , and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 , using Agrobacterium- mediated transfer into flowers.
  • transgene expression vectors pCambia 1201 -GPT including construct of SEQ ID NO: 27
  • pCambia 1201 -GS including construct of SEQ ID NO: 6
  • transgene expression vectors pCambia 1201 -GPT including construct of SEQ ID NO: 27
  • pCambia 1201 -GS including construct of SEQ ID NO: 6
  • transformed Agrobacterium were selected on media containing 50 ⁇ g/ml of chloroamphenicol.
  • Transformed Agrobacterium cells were grown in LB culture media containing 25 ⁇ g/ml of antibiotic for 36 hours. At the end of the 36 hr growth period cells were collected by centrifugation and cells from each transformation were resuspended in 100 ml LB broth without antibiotic.
  • Bean plants were then transformed with a mixture of the resulting Agrobacterium cell suspensions using a transformation protocol in which the Agrobacteria is injected directly into the flower structure (Yasseem, 2009, Plant Mol. Biol. Reporter 27: 20- 28).
  • a transformation protocol in which the Agrobacteria is injected directly into the flower structure.
  • 10 ⁇ g/ml acetosyringonone was added to the Agrobacteria cultures prior to flower inoculation. Briefly, once flowers bloomed, the outer structure encapsulating the reproductive organs was gently opened with forceps in order to permit the introduction of the Agrobacteria mixture, which was added to the flower structure sufficient to flood the anthers.
  • Glutamine synthetase (GS) activity was assayed according to the methods in Shapiro and Stadtmann, 1970, Methods in Enzymology 17A: 910- 922; and, Glutamine phenylpyruvate transaminase (GPT) activity was assayed according to the methods in Calderon et al., 1985, J. Bacteriol. 161 : 807-809. See details in Example 7, Methods, supra.
  • FIG. 10 shows GPT+GS transgenic bean line A growth rate data relative to control plants, including plant heights on various days into cultivation, as well as numbers of flower buds, flowers, and bean pods. These data show that the GPT+GS double transgenic bean plants outgrew their counterpart control plants. The transgenic plants grew taller, flowered earlier and produced more flower buds and flowers, and developed bean pods and produced more bean pods that the wild type control plants.
  • Table VI presents bean pod yield, GPT and GS activity, as well as antibiotic resistance status, in the transgenic lines compared to the wild type control (average of several robust control plants; control plants that did not grow well were excluded from the analyses).
  • double-transgene progeny plants showed substantial bean pod biomass increases (fresh pod weight) in comparison to the control plants, with bean pod biomass yields consistently above 200 grams per individual transgenic plant, compared to an average of 127 grams per wild type plant, representing an over 60% increase in pod yield in the double transgene lines relative to control plant(s).
  • FIG.1 1 shows a photograph of a GPT+GS double transgenic bean plant compared to a control plant grown for the same time under identical conditions, showing increased growth in the transgenic plant.
  • yellow wax bean plants ⁇ Phaseolus vulgaris were transformed with the Grape GPT full length coding sequence included in SEQ ID NO: 8 under the control of the RuBisCo promoter within the expression vector pCambia 1305.1 , and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 , using Agrobacterium-mediated transfer into developing pods.
  • transgene expression vectors pCambia 1201 -GPT(grape) including construct of SEQ ID NO: 8
  • pCambia 1201 -GS including construct of SEQ ID NO: 6
  • the transgene expression vectors pCambia 1201 -GPT(grape) including construct of SEQ ID NO: 8
  • pCambia 1201 -GS including construct of SEQ ID NO: 6
  • transgene expression vectors pCambia 1201 -GPT(grape) including construct of SEQ ID NO: 8
  • pCambia 1201 -GS including construct of SEQ ID NO: 6
  • Transformed Agrobacterium were selected on media containing 50 ⁇ g/ml of chloroamphenicol.
  • Transformed Agrobacterium cells were grown in LB culture media containing 25 ⁇ g/ml of antibiotic for 36 hours.
  • FIG. 12 shows GPT+GS transgenic bean line G growth rate data relative to control plants, specifically including numbers of flower buds, flowers, and bean pods. These data show that the GPT+GS double transgenic bean plants outgrew their counterpart control plants. Notably, the transgenic plants produced substantially more bean pods that the wild type control plants.
  • Table VII presents bean pod yield and antibiotic resistance status, in the transgenic lines compared to the wild type control (average of several robust control plants; control plants that did not grow well were excluded from the analyses).
  • double-transgene progeny plants showed substantial bean pod biomass increases (fresh pod weight) in comparison to the control plants, with bean pod biomass yields of 200.5 (line G1 ) and 178 grams (line G2) per individual transgenic plant, compared to an average of 158 grams per individual wild type plant, representing approximately a 27% increase in pod yield in the double transgene lines relative to control plants.
  • FIG. 13 shows a photograph of a GPT+GS double transgenic bean plant compared to a control plant grown for the same time under identical conditions.
  • the transgenic plant shows substantially increased size and biomass, larger leaves and a more mature flowering compared to the control plant.
  • EXAMPLE 11 GENERATION OF DOUBLE TRANSGENIC COWPEA PLANTS CARRYING ARABIDOPSIS GS1 AND GPT TRANSGENES:
  • FIG. 14 shows relative growth rates for the GPT+GS transgenic Cowpea line A and wild type control Cowpea at several intervals during cultivation, including (FIG. 14A) height and longest leaf measurements, (FIG. 14B) trifolate leafs and flower buds, and (FIG. 14C) flowers, flower buds and pea pods.
  • Table VIII presents pea pod yield, GPT and GS activity, as well as antibiotic resistance status, in the transgenic lines compared to the wild type control (average of several robust control plants; control plants that did not grow well were excluded from the analyses).
  • double-transgene progeny plants showed substantial pea pod biomass increases (fresh pod weight) in comparison to the control plants, with average transgenic plant pea pod biomass yields nearly 52% greater than the yields measured in control plant(s).
  • FIG. 15 shows a photograph of a GPT+GS double transgenic bean plant compared to a control plant grown for the same time under identical conditions, showing increased biomass and pod yield in the transgenic plant relative to the wild type control plant.
  • EXAMPLE 12 GENERATION OF DOUBLE TRANSGENIC COWPEA PLANTS CARRYING ARABIDOPSIS GS1 AND GRAPE GPT TRANSGENES:
  • FIG. 16 shows relative growth rates for the GPT+GS transgenic Cowpea line G and wild type control Cowpea. These data show that the transgenic plants are consistently higher (FIG. 16A), produce substantially more flowers, flower buds and pea pods (FIG. 16B), and develop trifolates and leaf buds faster (FIG. 16C).
  • Table IX presents pea pod yield, GPT and GS activity, as well as antibiotic resistance status, in the transgenic lines compared to the wild type control (average of several robust control plants; control plants that did not grow well were excluded from the analyses).
  • double-transgene progeny plants showed substantial pea pod biomass increases (fresh pod weight) in comparison to the control plants, with average pea pod biomass yields 70% greater in the transgenic plants compared to control plant(s).
  • FIG. 17 shows a photograph of a GPT+GS double transgenic pea plant compared to a control plant grown for the same time under identical conditions, showing increased height, biomass and leaf size in the transgenic plant relative to the wild type control plant.
  • EXAMPLE 13 GENERATION OF DOUBLE TRANSGENIC ALFALFA PLANTS CARRYING ARABIDOPSIS GS1 AND GPT TRANSGENES:
  • Alfalfa plants ⁇ Medicago sativa, var Ladak) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (including construct of SEQ ID NO: 6), using Agrobacterium-mediated transfer into seedling plants.
  • Agrobacterium vectors and mixtures were prepared for seedling inoculations as described in Example 1 1 , supra.
  • Alfalfa seedlings were still less than about 1 /2 inch tall, they were soaked in paper toweling that had been flooded with the Agrobacteria mixture containing both transgene constructs. The seedlings were left in the paper toweling for two to three days, removed and then planted in potting soil. Resulting TO and control plants were then grown for the first 30 days in a growth chamber, thereafter cultivated in a greenhouse, and then harvested 42 days after sprouting. At this point, only the transgenic Alfalfa line displayed flowers, as the wild type plants only displayed immature flower buds. The plants were characterized as to flowering status and total biomass.
  • Cantaloupe plants ⁇ Cucumis melo var common were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (including construct of SEQ ID NO: 6), using Agrobacterium-mediated transfer via injection into developing melons.
  • Agrobacterium vectors and mixtures were prepared for intra-melon inoculations as described in Example 8, supra. Inoculations into developing melons were carried out essentially as described in Example 8. The plants were characterized as to flowering status and total biomass relative to control melon plants grown under identical conditions.
  • the transgenic plants showed substantial foliar plant biomass increases in comparison to the control plants, with an average increase in biomass of 63%. Moreover, a tremendous increase in flower and flower bud yields was observed in all five transgenic lines. Control plants displayed no flowers and only 5 buds at sacrifice, on average. In sharp contrast, the transgenic plants displayed between 2 and 5 flowers per plant, and between 21 and 30 flower buds, per plant, indicating a substantially higher growth rate and flower yield. Increased flower yield would be expected to translate into correspondingly higher melon yields in the transgenic plants. Referring to FIG. 18 (a photograph comparing transgenic Cantaloupe plants to control Cantaloupe plants), the transgenic Cantaloupe plants show dramatically increased height, overall biomass and flowering status relative to the control plants.
  • EXAMPLE 15 GENERATION OF DOUBLE TRANSGENIC PUMPKIN PLANTS CARRYING ARABIDOPSIS GS1 AND GPT TRANSGENES:
  • common Pumpkin plants ⁇ Cucurbita maxima were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (including construct of SEQ ID NO: 6), using Agrobacterium-mediated transfer via injection into developing pumpkins, essentially as described in Example 14, supra.
  • the transgenic and control pumpkin plants were grown under identical conditions until the emergence of flower buds in the control plants, then all plants were characterized as to flowering status and total biomass.
  • the results are presented in FIG. 19 and Table XII.
  • the transgenic plants showed substantial foliar plant biomass increases in comparison to the control plants, with an increase in average biomass yield of 67% over control plants.
  • an increase in flower bud yields was observed in four of the five transgenic lines in comparison to control. Control plants displayed only 4 buds at sacrifice (average).
  • four transgenic plant lines displayed between 8 and 15 flowers buds per plant, representing a two- to nearly four-fold yield increase.
  • FIG. 19 a photograph comparing transgenic pumpkin plants to control plants
  • the transgenic pumpkin plants show substantially increased plant size, overall biomass and leaf sizes and numbers relative to the control plants.
  • EXAMPLE 16 GENERATION OF DOUBLE TRANSGENIC ARABIDOPSIS
  • Arabidopsis thaliana plants were transformed with the truncated Arabidopsis GPT coding sequence of SEQ ID NO: 18 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and transgenic plants thereafter transformed with the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (including construct of SEQ ID NO: 6), using Agrobacterium-mediated "floral dip" transfer as described (Harrison et al., 2006, Plant Methods 2:19-23; Clough and Bent, 1998, Plant J. 16:735-743).
  • Agrobacterium vectors pMON316 carrying GPT and pCambia 1201 carrying GS1 were prepared as described in Examples 3 and 1 1 , respectively. Transformation of two different cultures of Agrobacterium with either a pMon 316 + Arabidopsis GTP construct or with a Cambia 1201 + Arabidopsis GS construct was done by electroporation using the method of Weigel and Glazebrook 2002. The transformed Agrobacterium were then grown under antibiotic selection, collected by centrifugation resuspended in LB broth with antibiotic and used in the floral dip of Arabidopsis inflorescence. Floral dipped Arabidopsis plants were taken to maturity and self-fertilized and seeds were collected.
  • Seeds from twice dipped plants were first geminated on a media containing 20ug/ml of kanamycin and by following regular selection procedures surviving seedlings were transferred to media containing 20 ug of hygromycin. Plants (3) surviving the selection process on both antibiotics were self-fertilized and seeds were collected. Seeds from the T1 generation were germinated on MS media containing 20 ug/ml of hygromycin and surviving seedlings were taken to maturity, self-fertilized and seeds collected. This seed population the T2 generation was then used for subsequent growth studies. The results are presented in FIG. 20 and Table XIII.
  • Table XIII which shows data from 6 wild type and 6 transgenic Arabidopsis plants (averaged), the transgenic plants displayed increased levels of both GPT and GS activity. GPT activity was over twenty-fold higher than the control plants. Moreover, the transgenic plant fresh foliar weight average was well over four-fold that of the wild type control plant average. A photograph of young transgene Arabidopsis plants in comparison to wild type control Arabidopsis plants grown under identical conditions is shown in FIG. 20, and reveals a consistent and very significant growth/biomass increase in transgenic plants relative to the control plants.
  • EXAMPLE 17 GENERATION OF TRANSGENIC TOMATO PLANTS CARRYING ARABIDOPSIS GPT AND GS1 TRANSGENES:
  • tomato plants ⁇ Solatium lycopersicon, "Money Maker” variety) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, supra), and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 (including construct of SEQ ID NO: 6).
  • Single transgene (GPT) transgenic tomato plants were generated and grown to flowering essentially as described in Example 4.
  • the Arabidopsis GS1 transgene was then introduced into the single-transgene TO plants using Agrobacterium-mediated transfer via injection directly into flowers (as described in Example 8).
  • the transgenic and control tomato plants were grown under identical conditions and characterized as to growth phenotype characteristics. Resulting TO double-transgene plants were then grown to maturity, photographed along with control tomato plants, and phenotypically characterized.
  • the transgenic tomato plants displayed substantially larger leaves compared to control plants (FIG 21 A).
  • the transgenic tomato plants were substantially larger, taller and of a greater overall biomass (see FIG. 21 B).
  • EXAMPLE 18 GENERATION OF TRANSGENIC CAMILENA PLANTS CARRYING ARABIDOPSIS GPT AND GS1 TRANSGENES:
  • Camelina plants ⁇ Camelina sativa, Var MT 303) were transformed with the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1 under the control of the RuBisCo promoter within the expression vector pCambia 1201 , and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter within the expression vector pCambia 1201 , using Agrobacterium- mediated transfer into germinating seeds according to the method described in Chee et al., 1989, Plant Physiol. 91 : 1212-1218. Agrobacterium vectors and mixtures were prepared for seed inoculations as described in Example 1 1 , supra.
  • Transgenic and control Camelina plants were grown under identical conditions (30 days in a growth chamber and then moved to greenhouse cultivation) for 39 days, and characterized as to biomass, growth characteristics and flowering stage.
  • FIG. 22 shows a photograph of transgenic Camelina compared to control. The transgenic plant is noticeably larger and displays more advanced flowering.
  • the putative coding sequence for Barley GPT was isolated and expressed from a transgene construct using an in planta transient expression assay. Biologically active recombinant Barley GPT was produced, and catalyzed the increased synthesis of 2- oxoglutaramate, as confirmed by HPLC.
  • the Barley ⁇ Hordeum vulgare) GPT coding sequence was determined and synthesized.
  • the DNA sequence of the Barley GPT coding sequence used in this example is provided in SEQ ID NO: 14, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 15.
  • the coding sequence for Barley GPT was inserted into the 1305.1 cambia vector, and transferred to Agrobacterium tumefaciens strain LBA404 using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159), followed by plating on LB plates containing hygromycin (50 micro gm / ml). Antibiotic resistant colonies of Agrobacterium were selected for analysis.
  • the transient tobacco leaf expression assay consisted of injecting a suspension of transformed Agrobacterium (1 .5-2.0 OD 650) into rapidly growing tobacco leaves. Intradermal injections were made in a grid across the leaf surface to assure that a significant amount of the leaf surface would be exposed to the Agrobacterium. The plant was then allowed to grow for 3-5 days when the tissue was extracted as described for all other tissue extractions and the GPT activity measured.
  • the putative coding sequence for rice GPT was isolated and expressed in E. coli.
  • Biologically active recombinant rice GPT was produced, and catalyzed the increased synthesis of 2- oxoglutaramate, as confirmed by HPLC.
  • the rice ⁇ Oryza sativa) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in E. coli. Briefly, E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-B-D- thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25 X 106 cells were then assayed for biological activity using the NMR assay, below. Untransformed, wild type E. coli cells were assayed as a control. An additional control used E coli cells transformed with an empty vector.
  • the DNA sequence of the rice GPT coding sequence used in this example is provided in SEQ ID NO: 10, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 1 1 .
  • HPLC was used to determine 2-oxoglutaramate production in GPT-overexpressing E. coli cells, following a modification of Calderon et al., 1985, J Bacteriol 161 (2): 807- 809. Briefly, a modified extraction buffer consisting of 25 mM Tris-HCI pH 8.5, 1 mM EDTA, 20 ⁇ Pyridoxal phosphate, 10 mM Cysteine, and -1 .5% (v/v) Mercaptoethanol was used. Samples (lysate from E. coli cells, 25 X 106 cells) were added to the extraction buffer at approximately a 1 /3 ratio (w/v), incubated for 30 minutes at 37°C, and stopped with 200 ⁇ of 20% TCA.
  • the assay mixture is centrifuged and the supernatant used to quantify 2-oxoglutaramate by HPLC, using an ION-300 7.8mm ID X 30 cm L column, with a mobile phase in 0.01 N H2SO4, a flow rate of approximately 0.2 ml/min, at 40°C. Injection volume is approximately 20 ⁇ , and retention time between about 38 and 39 minutes. Detection is achieved with 210nm UV light.
  • the validation of the HPLC assay also included monitoring the disappearance of the substrate glutamine and showing that there was a 1 :1 molar stoechiometry between glutamine consumed to 2-oxoglutaramte produced.
  • the assay procedure always included two controls, one without the enzyme added and one without the glutamine added. The first shows that the production of the 2- oxoglutaramate was dependent upon having the enzyme present, and the second shows that the production of the 2-oxoglutaramate was dependent upon the substrate glutamine.
  • EXAMPLE 21 ISOLATION AND EXPRESSION OF RECOMBINANT SOYBEAN GPT GENE CODING SEQUENCE AND ANALYSIS OF BIOLOGICAL ACTIVITY
  • soybean GPT putative coding sequence for soybean GPT was isolated and expressed in E. coli.
  • Biologically active recombinant soybean GPT was produced, and catalyzed the increased synthesis of 2- oxoglutaramate, as confirmed by HPLC.
  • Soybean GPT coding sequence and expression in E. coli Soybean GPT coding sequence and expression in E. coli:
  • the soybean (Glycine max) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in E. coli. Briefly, E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-B-D- thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25 X 106 cells were then assayed for biological activity using the HPLC assay, below. Untransformed, wild type E. coli cells were assayed as a control. An additional control used E coli cells transformed with an empty vector.
  • the DNA sequence of the soybean GPT coding sequence used in this example is provided in SEQ ID NO: 12, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 13.
  • HPLC was used to determine 2-oxoglutaramate production in GPT-overexpressing E. coli cells, as described in Example 20, supra. Results:
  • soybean GPT coding sequence of SEQ ID NO: 12 resulted in the over-expression of recombinant GPT protein having 2-oxoglutaramate synthesis- catalyzing bioactivity. Specifically, 31 .9 nanomoles of 2-oxoglutaramate activity was observed in the E. coli cells overexpressing the recombinant soybean GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate activity in control E. coli cells, a nearly 1 , 600-fold activity level increase over control.
  • EXAMPLE 22 ISOLATION AND EXPRESSION OF RECOMBINANT ZEBRA FISH GPT GENE CODING SEQUENCE AND ANALYSIS OF BIOLOGICAL ACTIVITY
  • the putative coding sequence for Zebra fish GPT was isolated and expressed in E. coli.
  • Biologically active recombinant Zebra fish GPT was produced, and catalyzed the increased synthesis of 2- oxoglutaramate, as confirmed by HPLC.
  • Zebra fish GPT coding sequence and expression in E. coli Materials and Methods: Zebra fish GPT coding sequence and expression in E. coli:
  • the Zebra fish ⁇ Danio rend) GPT coding sequence was determined and synthesized, inserted into a PET28 vector, and expressed in E. coli. Briefly, E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-B-D- thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25 X 106 cells were then assayed for biological activity using the HPLC assay, below. Untransformed, wild type E. coli cells were assayed as a control. An additional control used E coli cells transformed with an empty vector.
  • the DNA sequence of the Zebra fish GPT coding sequence used in this example is provided in SEQ ID NO: 16, and the encoded GPT protein amino acid sequence is presented in SEQ ID NO: 17.
  • HPLC Assay for 2-oxoqlutaramate is provided in SEQ ID NO:
  • HPLC was used to determine 2-oxoglutaramate production in GPT-overexpressing E. coli cells, as described in Example 20, supra.
  • the DNA coding sequence of a truncation of the Arabidopsis thaliana GPT coding sequence of SEQ ID NO: 1 was designed, synthesized, inserted into a PET28 vector, and expressed in E. coli.
  • the DNA sequence of the truncated Arabidopsis GPT coding sequence used in this example is provided in SEQ ID NO: 20 (-45 AA construct), and the corresponding truncated GPT protein amino acid sequence is provided in SEQ ID NO: 21 .
  • E. coli cells were transformed with the expression vector and transformants grown overnight in LB broth diluted and grown to OD 0.4, expression induced with isopropyl-B-D-thiogalactoside (0.4 micromolar), grown for 3 hr and harvested. A total of 25 X 10 6 cells were then assayed for biological activity using HPLC as described in Example 20. Untransformed, wild type E. coli cells were assayed as a control. An additional control used E coli cells transformed with an empty vector
  • EXAMPLE 24 GPT + GS TRANSGENIC TOBACCO SEED GERMINATION TOLERATES HIGH SALT CONCENTRATIONS
  • seeds form the double transgene tobacco line XX-3 (Cross 3 in Table 4, see Example 7) were tested in a seed germination assay designed to evaluate tolerance to high salt concentrations.
  • Tobacco seeds from the wild type and XX-3 populations were surfaced sterilized (5% bleach solution for 5 minutes followed by a 10% ethanol wash for 3 minutes) and rinsed with sterile distilled water.
  • the surface sterilized seeds were then spread on Murashige and Skoog media (10% agarose) without sucrose and containing either 0 or 200 mM NaCI.
  • the seeds were allowed to germinate in darkness for 2 days followed by 6 days under a 16:8 photoperiod at 24°C. On day eight the rate of germination was determined by measuring the percentage of seeds from the control or transgene plants that had germinated.
  • Plant type Control (0 mM NaCI) Test (200 mM NaCI)a % Germination % Germination
  • EXAMPLE 25 METHOD FOR GENERATING TRANSGENIC MAIZE PLANTS CARRYING HORDEUM GPT AND GS1 TRANSGENES:
  • This example provides a method for generating transgenic maize plants expressing GPT and GS1 transgenes.
  • Maize ⁇ Zea mays, hybrid line Hi-ll) type II callus is biolistically transformed with an expression cassette including the hordeum glutamine synthetase (GS1 ) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo small subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SEQ ID NO: 44. Transformation of maize callus is achieved by particle bombardment.
  • An expression cassette including the hordeum GS1 and GPT genes, under the control of the rice RuBisCo small subunit and corn ubiquitin promoters, respectively, is cloned into the plasmid pAHC25 (Christensen and Quail, 1996, Transgenic Research 5:213-218) modified to include a bar gene conferring resistance to bialophos, or a similar vector, in order to generate the transgene expression vector.
  • the transgene expression vector is introduced into immature zygotic embryo source callus of parent maize hybrid line Hi-ll (A188xB73 origin) (Armstrong et al., 1991 , Maize Genetics Coop Newsletter 65:92-93) using particle bombardment, essentially as described (Frame et al., 2000, In Vitro Cell. Dev. Biol-Plant 36:21 -29; this method was developed by and is routinely used at the Iowa State University Center for Plant Transformation).
  • immature zygotic embryo source callus is prepared for transformation by serial culturing on a callus-initiating medium (N6E, Songstad et al., 1996, In vitro Cell Dev. Biol.- Plant 32:179-183). Washed gold particles are coated with the plasmid construct and used to bombard the callus with a PDS 1000/He biolistic gun as described (Sanford et al., 1993, Methods in Enzymology 217: 483- 509). After 7-10 days on initiation medium, the callus is then transferred to selection medium containing bialophos (N6S, Songstad et al., 1996, supra) and allowed to grow.
  • callus pieces are transferred to a regeneration medium (Armstrong and Green, 1985, Planta 164:207- 214) containing bialophos and allowed to grow for several weeks. Thereafter, the resulting plantlets are transferred to regeneration medium without the selection agent, and cultivated.
  • a regeneration medium Armstrong and Green, 1985, Planta 164:207- 2144 containing bialophos and allowed to grow for several weeks. Thereafter, the resulting plantlets are transferred to regeneration medium without the selection agent, and cultivated.
  • Transgenic corn plants may be grown and evaluated through maturity, and seeds harvested for use in generating subsequent generations of an event.
  • Various phenotypic characteristics may be observed in T 0 events, as well as in Ti and subsequent generations, and used to select seed sources for the development of subsequent generations.
  • High performing lines may be selfed to achieve trait homozygosity and/or crossed.
  • EXAMPLE 26 METHOD FOR GENERATING TRANSGENIC RICE PLANTS CARRYING HORDEUM GPT AND GS1 TRANSGENES:
  • This example provides a method for generating transgenic rice plants expressing GPT and GS1 transgenes.
  • Rice ⁇ Oryza sativa, Japonica cultivar Nipponbare) type II callus was transformed with the hordeum glutamine synthetase (GS1 ) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo small subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SEQ ID NO: 44. Transformation was achieved by Agrobacterium.
  • Base vector pTF101 .1 is a derivative of the pPZP binary vector (Hajdukiewicz et al 1994, Plant Mol. Biol. 25:989-994), which includes the right and left T-DNA border fragments from a nopaline strain of A.
  • the plant selectable marker gene cassette includes the phosphinothricin acetyl transferase ⁇ bar) gene from Streptomyces hygroscopicus that confers resistance to the herbicides glufosinate and bialophos.
  • the soybean vegetative storage protein terminator (Mason et al., 1993) follows the 3' end of the bar gene.
  • YEP Medium 5 g/L yeast extract, 10 g/L peptone, 5 g/L NaCI 2 , 15 g/L Bacto-agar. pH to 6.8 with NaOH. After autoclaving, the appropriate antibiotics were added to the medium when it has cooled to 50 °C.
  • Infection Medium N6 salts and vitamins (Chu et al., 1975, Sci. Sinica 18: 659-668), 1 .5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.7 g/L L-proline, 68.4 g/L sucrose, and 36 g/L glucose (pH 5.2).
  • This medium was filter-sterilized and stored at 4°C.
  • Acetosyringone (AS, 100 ⁇ ) was added just prior to use (prepared from 100 ⁇ stocks of filter-sterilized AS, dissolved in DMSO to 200mM then diluted 1 :1 with water).
  • N6 salts and vitamins 300 mg/L casamino acids, 2.8 g/L L- proline, 30 g/L sucrose, and 4 g/L gelrite (pH 5.8). Filter sterilized N6 Vitamins and 2 mg/L 2,4-D, were added to this medium after autoclaving.
  • Co-cultivation Medium make fresh: N6 salts and vitamins, 300 mg/L casamino acids, 30 g/L sucrose, 10 g/L glucose , and 4 g/L gelrite (pH 5.8). Filter sterilized N6 vitamins, acetosyringone (AS) 100 ⁇ and 2 mg/L 2,4-D were added to this medium after autoclaving.
  • N6 salts and vitamins 300 mg/L casamino acids, 30 g/L sucrose, 10 g/L glucose , and 4 g/L gelrite (pH 5.8).
  • Filter sterilized N6 vitamins, acetosyringone (AS) 100 ⁇ and 2 mg/L 2,4-D were added to this medium after autoclaving.
  • Selection Medium N6 salts and vitamins, 300 mg/L casamino acids, 2.8 g/L L-proline, 30 g/L sucrose, and 4 g/L gelrite (pH 5.8). Filter sterilized N6 vitamins, 2 mg/L 2,4-D, 2 mg/L Bialaphos (Shinyo Sangyo, Japan) and 500 mg/L carbenicillin were added to this medium after autoclaving.
  • Regeneration Medium I MS salts and vitamins (Murashige and Skoog, 1962), 2 g/L casamino acids, 30 g/L sucrose, 30 g/L sorbitol, and 4 g/L gelrite (pH 5.8). Filter sterilized MS vitamins, 100 mg/L cefotaxime, 100 mg/L vancomycin, 0.02 mg/L NAA (naphthaleneacetic acid), 2 mg/L kinetin (Toki, 1997, supra) and 2 mg/L Bialaphos were added to this medium after autoclaving.
  • MS vitamins 100 mg/L cefotaxime, 100 mg/L vancomycin, 0.02 mg/L NAA (naphthaleneacetic acid), 2 mg/L kinetin (Toki, 1997, supra) and 2 mg/L Bialaphos were added to this medium after autoclaving.
  • Regeneration Medium II MS Salts and vitamins, 100 mg/L myo-inositol, 30 g/L sucrose, 3 g/L gelrite, (pH 5.8).
  • callus tissue derived from the mature rice embryo was used as the starting material for transformation.
  • Callus induction, co-cultivation, selection and regeneration I media are based on those of Hiei et al., 1994, The Plant Journal 6 (2):271 -282.
  • calli were induced as follows. First, 15-20 rice seeds were dehusked and rinsed in 10 ml of 70% Ethanol (50 ml conical tube) by vigorously shaking the tube for one minute, followed by rinsing once with sterile water. Then, 10 ml of 50% commercial bleach (5.25 % hypochlorite) was added and placed on a shaker for 30 minutes (low setting). The bleach solution was then poured-off and the seeds rinsed five times with ⁇ 10ml of sterilized water each time. With a small portion of the final rinse, the seeds were poured onto sterilized filter paper (in a sterile petri plate) and then allowed to dry.
  • Ethanol 50 ml conical tube
  • the selection medium uses modifications from Toki (Toki, 1997, Plant Molecular Biology Reporter 15:16-21 ) whereby bialophos (2 mg/L) was employed for plant selection and carbenicillin (500 mg/L) for counter selection against Agrobacterium. Regeneration II medium was as described (Armstrong and Green, 1985, Planta 164:207-214).
  • rice calli were first placed into bacteria-free infection medium + AS (50 ml conical). This pre-wash was removed and replaced with 10 ml of the prepared Agrobacterium suspension (OD 550 ⁇ 0.1 ). Then, the conical was fastened onto a vortex shaker (low setting) for two minutes. After infection, calli were poured out of the conical onto a stack of sterile filter paper in a 100 x 15 petri dish to blot dry. Then, they were transferred off the filter paper and onto the surface of co-cultivation medium with sterile forceps. Co-cultivation plates were wrapped with vent tape and incubated in the dark at 25 Q C for three days.
  • the calli were washed five times with 5 ml of the liquid infection medium (no AS) supplemented with carbenicillin (500mg/L) and vancomycin (100mg/L). Calli were blotted dry on sterile filter paper as before. Individual callus pieces were transferred off the paper and onto selection medium containing 2 mg/L bialaphos. Selection plates were wrapped with parafilm and placed in the light at 29 °C.
  • liquid infection medium no AS
  • carbenicillin 500mg/L
  • vancomycin 100mg/L
  • plant tissue was cultured onto fresh selection medium every two weeks. This was done with the aid of a microscope to look for any evidence of Agrobacterium overgrowth. When overgrowth was noted, the affected calli were avoided (contaminated calli was not transferred). The remaining tissue was then carefully transferred, using newly sterilized forceps for each calli. Putative clones began to appear after six to eight weeks on selection. A clone was recognized as white, actively growing callus and was distinguishable from the brown, unhealthy non-transformed tissue. Individual transgenic events were identified and the white, actively growing tissue transferred to individual plates in order to produce enough tissue to take to regeneration.
  • Regeneration of transgenic plants was accomplished by selecting new lobes of growth from the callus tissue and transferring them onto Regeneration Medium I (light, 25 °C). After two to three weeks, the maturing tissue was transferred to Regeneration Medium II for germination (light, 25 °C). When the leaves were approximately 4-6 cm long and had developed good- sized roots, the plantlets were transferred (on an individual basis, typically 7-14 days after germination begins) to soilless mix using sterile conditions.
  • seedlings were transplanted into potting mix and adapted to ambient humidity and moderate light intensity for 3 days and then at 26°C under a photoperiod of 16 hr light and 8 hr dark in a controlled growth room designed by Conviron.
  • the seedlings were provided nutrient solution regularly; at first, application was once a week and then increased to twice and finally three times a week.
  • the nutrient solution contained 2.5 mM CaNO 3 , 2.5 mM KNO 3 , 1 mM MgSO 4 7H 2 O, 0.5mM KH 2 PO 4 , 0.5 mM H 3 SiO 3 , 0.045 mM H 3 BO 3 , 0.032 mM CuSO 4 , 0.071 mM ZnSO 4 , 0.01 1 mM MnCI 2 , 0.038 mM MoO 4 , Sequestrene 330 (70 mg/l) and set to pH 5.2.
  • the potting mix was kept wet by maintaining standing water/nutrient solution in the trays containing the small pots. Water was added as needed to maintain the desired high moisture content for the roots. Fourteen days after seedlings were transplanted to soil and adapted to ambient humidity, rice plants were photographed and sampled for biochemical and molecular analysis. Molecular Validation of Transgenic Plants by Standard PCR:
  • Genomic DNA was isolated from approximately 20mg plant leaf samples by using DNeasy Plant Mini Kit (Qiagen). Approximately 10-50ng of genomic DNA was used in Polymerase Chain Reactions (PCR). PCR was setup using Quick-Load Taq 2X Master Mix (New England Biolabs, final concentration of components: 10 mM Tris- HCI (pH 8.6), 50 mM KCI, 1 .5 mM MgCI 2 , 50 units/ml Taq DNA Polymerase, 0.2 mM each dNTP, 5% glycerol, 0.08% NP-40, 0.05% Tween-20, 0.024% Orange G, 0.0025% Xylene Cyanol FF, 200 nM each Forward and Reverse Primers).
  • PCR Polymerase Chain Reactions
  • GPT cassette upstream and downstream assays utilized, respectively, primers SV01 & SV02 and primers SV89 & SV108: SV01 5"- T AG C AG C ATCTTTG ACAAAGTCAGGGC-3' [SEQ ID NO: 61 ]
  • GPT and GS1 transgenes and the bar selectable marker gene in transformed rice plants was positively confirmed using standard PCR as described above.
  • GPT activity, GS activity, 2-oxoglutaramate concentration, and biomass of wild type and transgenic rice plants were measured and are shown in Table XXII below.
  • Transgenic rice plants produced, on average, nearly three times the biomass compared to wild type control rice plants, on average (183% increase over wild type rice).
  • the expression of the GPT transgene negatively impacted GPT expression in roots but resulted in elevated GPT activity in leaves.
  • the leaf:root ratio of 2-oxoglutaramate concentration was significantly elevated in the transgenic versus the control plants (increase of approximately 2-fold in the best growing plant).
  • FIG. 23 A photograph showing transgenic rice plants alongside wild type rice plants is shown in FIG. 23.
  • EXAMPLE 27 METHOD FOR GENERATING TRANSGENIC SUGARCANE PLANTS CARRYING HORDEUM GPT AND GS1 TRANSGENES:
  • This example provides a method for generating transgenic sugarcane plants expressing GPT and GS1 transgenes.
  • Sugarcane ⁇ Saccharum spp L) is biolistically transformed with an expression cassette including the hordeum glutamine synthetase (GS1 ) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo small subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SEQ ID NO: 44. Transformation of sugarcane callus is achieved by particle bombardment.
  • An expression cassette including the hordeum GS1 and GPT genes, under the control of the rice RuBisCo small subunit and corn ubiquitin promoters, respectively, are cloned into a small plasmid well established for sugarcane expression, such as pAHC20 (Thomson et al., 1987, EMBO J. 6:2519-2523), using standard molecular cloning methodologies, to generate the transgene expression vector.
  • the plasmid used contains a selectable marker against either the phospinothricin family of herbicides or the antibiotics geneticin or kanamycin, each of which have been shown effective (Ingelbrecht et al., 1999, Plant Physiology 1 19:1 187-1 197; Gallo-Maegher & Irvine, 1996, Crop Science 36:1367-1374).
  • the plasmid containing the expression cassette encoding the hordeum GS1 and GPT coding sequences is introduced into embryogenic callus prepared for transformation by the basic method of Gallo-Maegher and Irvine (Gallo-Maegher and Irvine, 1996, supra) and Ingelbrecht et al. (Ingelbrecht et al., 1999, supra) with the improved stimulation of shoot regeneration with thidiazuron (Gallo-Maegher et al., 2000, In vitro Cell Dev. Biol. - Plant 36:37-40).
  • This particle bombardment method is effective in transforming sugarcane (see, for example, Gilbert et al., 2005, Crop Science 45:2060-2067; and see the foregoing references).
  • Regenerable sugarcane varieties such as the commercial varieties CP65-357 and CP72-1210, may be used to generate transgene events. Briefly, 7- to 40-week old calli are bombarded with plasmid-coated tungsten or gold particles. Two days after bombardment the calli are transferred to selection medium. Four weeks later the resistant calli are transferred to shoot-induction medium containing the selection agent and sub-cultured every two weeks for approximately 12 weeks, at which time the shoots are transferred to Magenta boxes containing rooting medium with selection agent. The shoots are maintained on this medium for approximately 8 weeks, at which time those with good root development are transferred to potting mix and the adapted to atmospheric growth.
  • Transgenic sugarcane plants may be grown and evaluated through maturity, and seeds harvested for use in generating subsequent generations of an event.
  • Various phenotypic characteristics may be observed in T 0 events, as well as in Ti and subsequent generations, and used to select seed sources for the development of subsequent generations.
  • High performing lines may be selfed to achieve trait homozygosity and/or crossed.
  • EXAMPLE 28 METHOD FOR GENERATING TRANSGENIC WHEAT PLANTS CARRYING HORDEUM GPT AND GS1 TRANSGENES: This example provides a method for generating transgenic wheat plants expressing GPT and GS1 transgenes.
  • Wheat ⁇ Triticum spp. is biolistically transformed with an expression cassette including the hordeum glutamine synthetase (GS1 ) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo small subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NNO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SEQ ID NO: 44. Transformation of wheat callus is achieved by particle bombardment.
  • GS1 hordeum glutamine synthetase
  • SEQ ID NO: 40 under the control of the rice RuBisCo small subunit promoter of SEQ ID NO: 39
  • the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SEQ ID NO: 44. Transformation of wheat callus is achieved by particle bombardment.
  • An expression cassette including the hordeum GS1 and GPT genes, under the control of the rice RuBisCo small subunit and corn (maize) ubiquitin promoters, respectively, are cloned into a plasmid such as pAHC17, which contains the bar gene to provide the desired resistance to the phosphinothricin- class of herbicides for selection of transformants, using standard molecular cloning methodologies, to generate the transgene expression vector.
  • Bombarded embryos are cultured without selection for 1 -3 weeks in the dark on MS media before transferring them to shoot induction medium (MS media plus hormones and selection agent bialophos (1 ,1 .5, 2, 3 mg/L) for 2-8 weeks with subculturing weekly (Blechl et al., 2007, J Cereal Science 45:172-183).
  • Shoots that formed are transferred to rooting medium also containing the selection agent (bialophos 3 mg/L) (Weeks et al., 1993, supra).
  • Well-rooted plantlets are transferred to potting media and adapted to atmospheric growth conditions.
  • Transgenic wheat plants may be grown and evaluated through maturity, and seeds harvested for use in generating subsequent generations of an event.
  • Various phenotypic characteristics may be observed in T 0 events, as well as in Ti and subsequent generations, and used to select seed sources for the development of subsequent generations.
  • High performing lines may be selfed to achieve trait homozygosity and/or crossed.
  • EXAMPLE 29 METHOD FOR GENERATING TRANSGENIC SORGHUM PLANTS CARRYING HORDEUM GPT AND GS1 TRANSGENES:
  • This example provides a method for generating transgenic sorghum plants expressing GPT and GS1 transgenes.
  • Sorghum ⁇ Sorghum spp L is transformed with Agrobacterium carrying an expression cassette encoding the hordeum glutamine synthetase (GS1 ) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SE ID NO: 44.
  • GS1 hordeum glutamine synthetase
  • An expression cassette including the hordeum GS1 and GPT genes, under the control of the rice RuBisCo small subunit and corn ubiquitin promoters, respectively, is cloned into a stable binary vector such as pZY101 (Vega et al 2008, Plant Cell Rep. 27:297-305), using standard molecular cloning methodologies, to generate the transgene expression vector. Transformation and Regeneration:
  • Agrobacterium-mediated transformation and recovery of transgenic sorghum plants is as described (Lu et al., 2009, Plant Cell Tissue Organ Culture 99:97-108). These methods are routinely used by the University of Missouri Plant Transformation Core Facility.
  • the public sorghum line, P898012 is grown as described (Lu et al., 2009, supra) and transformed with Agrobacterium tumefaciens strain EHA101 (Hood et al., 1986, supra) transformed with the transgene expression vector.
  • Agrobacterium (0.3-0.4 OD) harboring the transgene expression vector is used to inoculate immature sorghum embryos for 5 minutes. The embryos are then transferred onto filter paper on top of their co-cultivation medium, containing acetosyringone to enhance the effectiveness of the infection. Embryos are incubated for 3-5 days and then transferred for another 4 days on resting medium (containing carbenicillin) and then transferred onto callus induction medium (with selection agent PPT) with weekly transfers. Once somatic embyrogenic cells develop they are transferred onto shooting medium (with carbenicillin and PPT) until shoots (2-5 cm long) develop. Shoots are transferred to Magenta boxes with rooting medium (with PPT) and maintained in 16 h light and 8 h darkness until 8-20 cm tall well-rooted plantlets are produced. They are then transferred to potting mix and adapted to atmospheric conditions.
  • Transgenic sorghum plants may be grown and evaluated through maturity, and seeds harvested for use in generating subsequent generations of an event.
  • Various phenotypic characteristics may be observed in T 0 events, as well as in Ti and subsequent generations, and used to select seed sources for the development of subsequent generations.
  • High performing lines may be selfed to achieve trait homozygosity and/or crossed.
  • EXAMPLE 30 METHOD FOR GENERATING TRANSGENIC SWITCHGRASS PLANTS CARRYING HORDEUM GPT AND GS1 TRANSGENES:
  • This example provides a method for generating transgenic switchgrass plants expressing GPT and GS1 transgenes.
  • Switchgrass ⁇ Panicum virgatum is transformed with Agrobacterium carrying a transgene expression vector including an expression cassette encoding the hordeum glutamine synthetase (GS1 ) coding sequence of SEQ ID NO: 40 under the control of the rice RuBisCo small subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil) promoter of SE ID NO: 44.
  • Agrobacterium-mediated transformation and recovery of transgenic switchgrass plants is essentially as described (Somleva et al., 2002, Crop Science 42:2080-2087; Somleva 2006, Switchgrass ⁇ Panicum virgatum L.) In Methods in Molecular Biology Vol 344. Agrobacterium Protocols 2/e, Volume 2. Ed K. Wang Humana Press Inc., Totowa, NJ.; Xi et al 2009, Bioengineering Research 2:275-283). These methods are routinely used by the Plant Biotechnology Resource and Outreach Center at Michigan State University. Briefly, explants of embryonic callus from the mature caryopses of the public Switchgrass cv.
  • Alamo are transformed with Agrobacterium tumefaciens strain EHA105 (Hood et al., 1986, supra) carrying the transgene expression vector.
  • Agrobacterium (0.8-1 .0 OD) harboring the transgene expression vector and pretreated with acetosynringone is used to inoculate the switchgrass callus for 10 minutes and then co-cultivated for 4-6 days in the dark.
  • the explants are then washed free of the agrobacterium and placed on selection medium containing the antibiotic timentin and hygromycin; selection requires 2-6 months. Subculturing is carried out at 4-week intervals.
  • Regeneration is accomplished in 4-8 weeks on media containing GA3, timentin and hygromycin under a photoperiod of 16 h light and 8 dark.
  • the plantlets are then transferred to Magenta boxes with regeneration medium containing GA3, timentin and hygromycin for another 4 weeks as before.
  • the plants are then transferred to soil and adapted to atmospheric growth.
  • Transgenic switchgrass plants may be grown and evaluated through maturity, and seeds harvested for use in generating subsequent generations of an event.
  • Various phenotypic characteristics may be observed in T 0 events, as well as in Ti and subsequent generations, and used to select seed sources for the development of subsequent generations.
  • High performing lines may be selfed to achieve trait homozygosity and/or crossed.
  • EXAMPLE 31 METHOD FOR GENERATING TRANSGENIC SOYBEAN PLANTS CARRYING ARABIDOPSIS GPT AND GS1 TRANSGENES: This example provides a method for generating transgenic soybean plants expressing GPT and GS1 transgenes.
  • Soybean ⁇ Glycine max was transformed with Agrobacterium carrying a transgene expression vector including an expression cassette encoding the Arabidopsis glutamine synthetase (GS1 ) coding sequence of SEQ ID NO: 7 under the control of the tomato RuBisCo small subunit promoter of SEQ ID NO: 22 (expression cassette of SEQ ID NO: 47), and the Arabidopsis GPT coding sequence of SEQ ID NO: 1 under the control of the 35S cauliflower mosaic virus (CMV) promoter (expression cassette of SEQ ID NO: 27).
  • GS1 Arabidopsis glutamine synthetase
  • CMV 35S cauliflower mosaic virus
  • pTF101 .1 is a derivative of the pPZP binary vector (Hajdukiewicz et al 1994, Plant Mol. Biol. 25:989-994), which includes the right and left T-DNA border fragments from a nopaline strain of A.
  • the plant selectable marker gene cassette includes the phosphinothricin acetyl transferase ⁇ bar) gene from Streptomyces hygroscopicus that confers resistance to the herbicides glufosinate and bialophos.
  • the soybean vegetative storage protein terminator (Mason et al., 1993) follows the 3' end of the bar gene.
  • YEP Solid Medium 5 g/L Yeast extract, 10 g/L Peptone, 5 g/L NaCI 2 , 12 g/L Bacto- agar. pH to 7.0 with NaOH. Appropriate antibiotics are added to the medium after autoclaving. Pour into sterile 100x15 plates ( ⁇ 25ml per plate).
  • YEP Liquid Medium 5 g/L Yeast extract, 10 g/L Peptone, 5 g/L NaCI 2 . pH to 7.0 with NaOH. Appropriate antibiotics are added to the medium prior to inoculation.
  • Co-cultivation Medium 1 /1 OX B5 major salts, 1 /1 OX B5 minor salts, 2.8 mg/L Ferrous, 3.8 mg/L NaEDTA, 30 g/L Sucrose, 3.9 g/L MES, and 4.25 g/L Noble agar (pH 5.4).
  • Infection Medium 1 /1 OX B5 major salts, 1 /1 OX B5 minor salts, 2.8 mg/L Ferrous, 3.8 mg/L NaEDTA, 30 g/L Sucrose, 3.9 g/L MES (pH 5.4).
  • Filter sterilized 1 X B5 vitamins, GA3 (0.25 mg/L), BAP (1 .67 mg/L), and 40 mg/L acetosyringone are added to this medium after autoclaving.
  • Shoot Induction Washing Medium 1 X B5 major salts, 1 X B5 minor salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 30 g/L Sucrose, and 0.59 g/L MES (pH 5.7).
  • Rooting Medium 1 X MS major salts, 1 X MS minor salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 20 g/L Sucrose, 0.59 g/L MES, and 7 g/L Noble agar (pH 5.6). Filter sterilized 1 X B5 vitamins, Asparagine (50 mg/L), and L-Pyroglutamic Acid (100 mg/L) are added to this medium after autoclaving. Media was poured into sterile 150x25 mm vial (10ml/vial).
  • Agrobacterium cultures were prepared for infecting seed explants as follows.
  • the vector system, pTF101 .1 in EHA101 was cultured on YEP medium (An et al., 1988) containing 100 mg/L spectinomycin (for pTF101 .1 ), 50 mg/L kanamycin (for EHA101 ), and 25 mg/L chloramphenicol (for EHA101 ).
  • a 2 ml culture of Agrobacterium was started by inoculating a loop of bacteria from the fresh YEP plate in YEP liquid medium amended with antibiotics. This culture was allowed to grow to saturation (8-10 hours) at 28 ° C in a shaker incubator (-250 rpm).
  • starter culture was transferred to a 1 L flask containing 250 ml of YEP medium amended with antibiotics.
  • Agrobacteria-containing infection medium was shaken at 60 rpm for at least 30 minutes before use.
  • Explants were prepared for inoculation as follows. Seeds were sterilized with a combination of bleach solution and exposure to chlorine gas. Prior to infection, (-20 hours), sees were imbibed with deionized sterile water in the dark. Imbibed soybean seeds were transferred to a sterile 100x15 petri plate for dissection. Using a scalpel (i.e., #15 blade), longitudinal cuts are made along the hilum to separate the cotyledons and remove the seed coat. The embryonic axis found at the nodal end of the cotyledons was excised, and any remaining axial shoots/buds attached to the cotyledonary node were also removed.
  • a scalpel i.e., #15 blade
  • Agrobacterium-mediated transformation was conducted as follows. Half-seed explants were dissected into a 100 x 25 mm petri plate and 30 ml Agrobacterium- containing infection media added thereto, such that the explants were completely covered by the infection media. Explants were allowed to incubate at room temperature for a short period of time (i.e., 30 minutes), with occasional gentle agitation.
  • the explants were transferred to co-cultivation medium, so that the flat, axial side was touching the filter paper. These plates were wrapped in parafilm, and cultivated for 5 days at 24°C under an 18:6 photoperiod. Following this co- cultivation, shoot growth was induced by first washing the explants in shoot induction washing medium at room temperature, followed by placing the explants in shoot induction medium I, such that the explants were oriented with the nodal end of the cotyledon imbedded in the medium and the regeneration region flush to the surface with flat side up (typically at a 30-45° angle). Explants were incubated at 24°C, 18:6 photoperiod, for 14 days. Explants were thereafter transferred to shoot induction medium II and maintained under the same conditions for another 14 days.
  • explants were transferred to shoot elongation medium, as follows. First, cotyledons ere removed from the explants. A fresh cut at the base of the shoot pad flush to the medium was made, and the explants transferred to shoot elongation medium (containing glufosinate) and incubated at 24 °C, 18:6 photoperiod, for 2-8 weeks. Typically, explant tissue was transferred to fresh shoot elongation medium every 2 weeks, and at transfer, a fresh horizontal slice at the base of the shoot pad was made.
  • shoot elongation medium containing glufosinate
  • shoots surviving the glufosinate selection reached ⁇ 3 cm length, they were excised from the shoot pad, briefly dipped in indole-3-butyric acid (1 mg/ml, 1 -2 minutes), then transferred to rooting medium for acclimatization (i.e., in 150 x 25 mm glass vials with the stems of the shoots embedded approximately 1 /2 cm into the media).
  • rooting medium i.e., in 150 x 25 mm glass vials with the stems of the shoots embedded approximately 1 /2 cm into the media.
  • the shoots were transferred to soil and plantlets grown at 24°C, 18:6 photoperiod, for at least one week, watering as needed.
  • Plants are provided a steady supply of a complete nutrient solution containing 0.5 mM KH 2 PO 4 , 1 mM MgSO 4 , 0.125 mM K 2 SO 4 , 7.5 mM KNO 3 , 2.6 mM CaCI 2 and 26mg/L sequestrene (10% Fe.).
  • a complete nutrient solution containing 0.5 mM KH 2 PO 4 , 1 mM MgSO 4 , 0.125 mM K 2 SO 4 , 7.5 mM KNO 3 , 2.6 mM CaCI 2 and 26mg/L sequestrene (10% Fe.).
  • Relative Growth Rate was determined within a hydroponic culture system, in which plants previously grown in soil were transferred to and grown in an aerated solution culture at 24°C, with a photoperiod of 16 hr light and 8 hr dark.
  • the nutrient media used to feed the plants in the hydroponic culture system was the same as the solution used to feed the plants in soil culture, supra.
  • Instantaneous RGR was calculated using a previously described method (South, D., 1993, Relative growth rates: A critique. South African Forestry J. 173:43-48).
  • Genomic DNA was isolated from approximately 20mg plant leaf samples by using DNeasy Plant Mini Kit (Qiagen). Approximately 10-50ng of genomic DNA was used in Polymerase Chain Reactions (PCR). PCR was setup using Quick-Load Taq 2X Master Mix (New England Biolabs, final concentration of components: 10 mM Tris- HCI (pH 8.6), 50 mM KCI, 1 .5 mM MgCI 2 , 50 units/ml Taq DNA Polymerase, 0.2 mM each dNTP, 5% glycerol, 0.08% NP-40, 0.05% Tween-20, 0.024% Orange G, 0.0025% Xylene Cyanol FF, 200 nM each Forward and Reverse Primers).
  • PCR Polymerase Chain Reactions
  • GPT and GS1 expression cassettes were conducted utilizing PCR assays directed to the promoter/CDS junction and CDS/terminator junction.
  • a downstream assay was utilized with the following primer pair: SV1 12 5"-CACAACCCGACCGGGAAGATGT-3" [SEQ ID NO: 65]
  • SV1 13 5'-GCAAGACCGGCAACAGGATTCA-3' [SEQ ID NO: 66]
  • upstream and downstream assays utilized, respectively, primers SV093 & SV094 and primers SV092 & SV108, below.
  • GPT activity , GS activity, 2-oxoglutaramate concentration, and RGRs relative growth rates of wild type and transgenic soybean plants were measured and are shown in Table XXIII below. Dramatic increases across all parameters measured were observed in the transgenic soybean versus wild type soybean plants. More specifically, leaf GPT activity increased by 7-fold, leaf GS activity more than doubled, 2-oxoglutaramate concentration in leaf tissue increasing by over 8-fold, and RGR increased by 67% in the GS+GPT transgenic soybean relative to wild type soybean plants.
  • EXAMPLE 32 METHOD FOR GENERATING TRANSGENIC POTATO PLANTS CARRYING ARABIDOPSIS GPT AND GS1 TRANSGENES:
  • This example provides a method for generating transgenic potato plants expressing GPT and GS1 transgenes.
  • Potato ⁇ Solatium tuberosum, cultivar Desiree is transformed with Agrobacterium carrying a transgene expression vector including an expression cassette encoding the Arabidopsis glutamine synthetase (GS1 ) coding sequence of SEQ ID NO: 7 under the control of the tomato RuBisCo small subunit promoter of SEQ ID NO: 22 (expression cassette of SEQ ID NO: 47), and the Arabidopsis GPT coding sequence of SEQ ID NO: 1 under the control of the 35S cauliflower mosaic virus (CMV) promoter (expression cassette of SEQ ID NO: 27).
  • CMV cauliflower mosaic virus
  • An expression cassette including the hordeum GS1 and GPT genes, under the control of the tomato RuBisCo small subunit and 35S CMV promoters, respectively, is cloned into the Cambia 2201 vector which provides kanamycin resistance.
  • a suitable Agrobacterium tumefaciens strain such as UC-Riverside Agro-1 strain is employed and used for infecting potato explant tissue (see, Narvaez-Vasquez et al., 1992, Plant Mo. Biol. 20:1 149-1 157). Cultures are maintained at 28°C in liquid medium containing 10 g/L Yeast extract, 10 g/L Peptone, 5 g/L NaCI 2 , 10 mg/L kanamycin, 30 mg/L tetracycline, and 9.81 g/L Acetosyringone (50 mM).
  • Potato leaf discs or tuber discs may be used as the explants to be inoculated.
  • Discs are pre-conditioned by incubation on feeder plates for two to three days at 25°C under dark conditions.
  • Pre-conditioned explants are infected with Agrobacterium by soaking in 20 ml of sterile liquid MS medium ⁇ supra), containing 10 8 Agrobacterium cells/ml for about 20 minutes.
  • the explants are carefully punched with a syringe needle, or scalpel blade. Then, the explants are blotted dry with sterile filter paper, and incubated again in feeder plates for another two days.
  • Explants are then transferred to liquid medium with transgene-transformed Agrobacterium, and incubated for three days at 28°C under dark conditions for calli and shoot development (development (2-4 cm) in the presence of kanamycin (100 mg/L). Following co-cultivation, supra, the explants are washed three times with sterile liquid medium and finally rinsed with the same medium containing 500 mg/l of cefotaxime.
  • the explants are blotted dry with sterile filter paper and placed on shoot induction medium (4.3 g/L MS salts, 10 mg/L thiamine, 1 mg/L nicotinic acid, 1 mg/L pyridxine, 100 mg/L inositol, 30 g/L sucrose, 1 mg/L zeatin, 0.5 mg/L IAA, 7 g/L phytoagar, 250 mg/L Cefotaxime, 500 mg/L Carbenicillin, 100 mg/L Kanamycin) for 4-6 weeks.
  • plantlets are transferred to rooting medium (4.3 g/L MS salts, 10 mg/L thiamine, 1 mg/L nicotinic acid, 1 mg/L pyridxine, 100 mg/L inositol, 20 g/L sucrose, 50 ⁇ g/L IAA, 7 g/L phytoagar, 50 mg/L Kanamycin and 500 mg/L Vancomycin) for 3-4 weeks.
  • rooting medium 4.3 g/L MS salts, 10 mg/L thiamine, 1 mg/L nicotinic acid, 1 mg/L pyridxine, 100 mg/L inositol, 20 g/L sucrose, 50 ⁇ g/L IAA, 7 g/L phytoagar, 50 mg/L Kanamycin and 500 mg/L Vancomycin
  • SEQ ID NO: 1 Arabidopsis glutamine phenylpyruvate transaminase DNA coding sequence:
  • SEQ ID NO: 6 Arabidopsis GS1 coding sequence
  • Cambia 1201 vector + rbcS3C+arabidopsis GS1 Bold ATG is the start site, AAAAAAGAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGG ACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCG TTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTA ACCAATTATTTCAGCACCdTGTCTCTGCTCTCAGATCTCGTTAACCTCAACCTCA CCGATGCCACCGGGAAAATCATCGCCGAATACATATGGATCGGTGGATCTGGA ATGGATATCAGAAGCAAAGCCAGGACACTACCAGGACCAGTGACTGATCCATCA AAGCTTCCCAAGTGGAACTACGACGGATCCAGCACCGGTCAGGCTGCTGGAGA AGACAGTGAAGTCATTCTATACCCTCAGG
  • a AG CCTTATC ACTATATATAC AAG G
  • Rice GPT codon optimized for E. coli expression untranslated sequences shown in lower case atgtggATGAACCTGGCAGGCTTTCTGGCAACCCCGGCAACCGCAACCGCAACCC
  • Vector sequences are italicized GCA TCA TCACCA TCACCATGGTAAGCCTA TCCCTAACCCTCTCCTCGGTCTC GA TTC TA CGGAAAACCTG TA TTTTCAGGGAA TTGA TCCCTTCA CCG CG AA ACGT CTGGAAAAATTTCAGACCACCATTTTTACCCAGATGAGCCTGCTGGCGATTAAAC ATGGCGCGATTAACCTGGGCCAGGGCTTTCCGAACTTTGATGGCCCGGAATTT GTGAAAGAAGCGGCGATTCAGGCGATTCGTGATGGCAAAAACCAGTATGCGCG TGGCTATGGCGTGCCGGATCTGAACATTGCGATTGCGGAACGTTTTAAAAAAGA TACCGGCCTGGTGGTGGATCCGGAAAAAGAAATTACCGTGACCAGCGGCTGCA CCGAAGCGATTGCGGCGACCATGATTGGCCTGATTAACCCGGGCGATGAAGTG ATTATGTTTGCGCCGTTTTATGATAGCTATGAAGCGACCCTGAG
  • SEQ ID NO: 21 Arabidopsis truncated GPT -45 construct amino acid sequence
  • SEQ ID NO: 23 bamboo GPT DNA coding sequence
  • SEQ ID NO: 25 1305.1 +rbcS3C promoter + catl intron with rice GPT gene.
  • SEQ ID NO: 27 Expression cassette, Arabidopsis GPT coding sequence (ATG underlined) under control of CMV 35S promoter (italics; promoter from Cambia 1201 )
  • SEQ ID NO: 28 Cambia p1305.1 with (3' end of) rbcS3C+Arabidopsis GPT coding sequence. Underlined ATG is start site, parentheses are the catl intron and the underlined actagt is the spel cloning site used to splice in the Arabidopsis gene.
  • SEQ ID NO: 33 Soybean GPT amino acid sequence (-1 mature protein, no targeting sequence) AKRLEKFQTTIFTQMSLLAIKHGAINLGQGFPNFDGPEFVKEAAIQAIRDGKNQYARG YGVPDLNIAIAERFKKDTGLVVDPEKEITVTSGCTEAIAATMIGLINPGDEVIMFAPFY DSYEATLSMAGAKVKGITLRPPDFAVPLEELKSTISKNTRAILINTPHNPTGKMFTRE ELNCIASLCIENDVLVFTDEVYDKLAFDMEHISMASLPGMFERTVTLNSLGKTFSLTG WKIGWAIAPPHLSWGVRQAHAFLTFATAHPFQCAAAAALRAPDSYYVELKRDYMAK RAILIEGLKAVGFKVFPSSGTYFVVVDHTPFGLENDVAFCEYLVKEVGVVAIPTSVFY LNPEEGKNLVRFTFCKDEETIRSAVERMKAKLRKVD SEQ ID NO: 34 Barley GPT amino acid sequence
  • SEQ ID NO: 35 Zebra fish GPT amino acid sequence (mature protein, no targeting sequence) VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAIRDGNNQYA RGYGVPDLNIAISERYKKDTGLAVDPEKEITVTSGCTEAIAATVLGLINPGDEVIVFAP FYDSYEATLSMAGAKVKGITLRPPDFALPIEELKSTISKNTRAILLNTPHNPTGKMFTP EELNTIASLCIENDVLVFSDEVYDKLAFDMEHISIASLPGMFERTVTMNSLGKTFSLT GWKIGWAIAPPHLTWGVRQAHAFLTFATSNPMQWAAAVALRAPDSYYTELKRDYM AKRSILVEGLKAVGFKVFPSSGTYFVVVDHTPFGHENDIAFCEYLVKEVGVVAIPTSV FYLNPEEGKNLVRFTFCKDEGTLRAAVDRMKEKLRK
  • SEQ ID NO: 38 Primer sequence, see Example 1 .
  • SEQ ID NO: 39 Rice rubisco promoter deposited in NCBI GenBank: AF143510.1
  • the construct also includes a Pmll 1305.1 cloning site CACGTG (also cuts in rice rbsc promoter), and a Zral cloning site GACGTC, which can be added by PCR to clone into Pmll site of vector).
  • SEQ ID NO: 42 Expression cassette combining SEQ ID NO: 39 (5') and SEQ ID NO: 40 (3'), encoding the Rice rubisco promoter, catl intron and part of Gus plus protein, and hordeum GS1 .
  • SEQ ID NO: 39 Hordeum GS1 coding sequence begins after Spel cloning site (double underline).
  • SEQ ID NO: 43 Amino acid sequence of translation product of SEQ ID NO: 42. Amino-terminal bold residues from Gusplus and Spel cloning site (intron removed)
  • SEQ ID NO: 47 Tomato rubisco small subunit (rbcS3C) promoter + Arabidopsis GS1 DNA coding sequence; Ncol/Afllll splice site shown in bold, ATG start of GS1 underlined.
  • SEQ ID NO: 49 Putative Clementine orange GPT amino acid sequence; putative mature protein sequence begins at VAK shown in bold underline. MLKPSAFGSSFSSSALLSFSKHLHTISITDSVNTRRRGISTACPRYPSLMASLSTVST
  • SEQ ID NO: 52 Maize ⁇ Zea mays) GPT amino acid sequence, see FIG 2.
  • SEQ ID NO: 53 Cotton ⁇ Gossypium hirsutum) GPT amino acid sequence, see FIG. 2.
  • SEQ ID NO: 54 Castor oil plant ⁇ Ricinus communis) GPT amino acid sequence, see FIG. 2.
  • SEQ ID NO: 55 California poplar ⁇ Populus trichocarpa) GPT amino acid sequence, see FIG. 2.
  • SEQ ID NO: 56 Soybean ⁇ Glycine max) GPT amino acid sequence, see FIG. 2.
  • SEQ ID NO: 57 Bryophyte moss ⁇ Physcomitrella patens) GPT amino acid sequence, see FIG. 2.
  • SEQ ID NO: 58 Green algae ⁇ Chlamydomonas sp.) GPT amino acid sequence, see FIG. 2.
  • SEQ ID NO: 59 GPT consensus amino acid sequence, see FIG. 2.
  • SEQ ID NO: 60 GPT consensus amino acid sequence (GPT subset), see FIG. 3.
  • SEQ ID NO: 61 Primer sequence, see Example 26.
  • SEQ ID NO: 62 Primer sequence, see Example 26.
  • SEQ ID NO: 63 Primer sequence, see Example 26.
  • SEQ ID NO: 64 Primer sequence, see Example 26.
  • SEQ ID NO: 65 Primer sequence, see Example 31 .
  • SEQ ID NO: 66 Primer sequence, see Example 31 .
  • SEQ ID NO: 67 Primer sequence, see Example 31 .
  • SEQ ID NO: 68 Primer sequence, see Example 31 .
  • SEQ ID NO: 69 Primer sequence, see Example 31 .
  • SEQ ID NO: 70 Primer sequence, see Example 31 .

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Abstract

La présente invention concerne des plants de riz et de soja transgéniques qui présentent des taux de croissance considérablement améliorés, des rendements supérieurs des graines, une utilisation plus efficace et plus précoce de l'azote, une tolérance accrue aux fortes teneurs en sel et des rendements accrus de la biomasse. Les plants transgéniques de l'invention sont conçus pour produire en excédent de la glutamine phénylpyruvate transaminase (GPT) ou à la fois de la glutamine phénylpyruvate transaminase et de la glutamine synthétase (GS). L'invention concerne également des protéines GPT de riz et de soja codant des nucléotides purifiés. Les plants transgéniques GPT et GPT+GS de l'invention montrent, de façon homogène, des caractéristiques de croissance améliorées, les lignes de génération TO montrant une augmentation de la biomasse par rapport à des équivalents du type sauvage, comprise entre 50 % et 300 %. Les générations issues des croisements sexuels et/ou de l'autofécondation se comportent encore mieux, certains des plants doublement transgéniques parvenant à une étonnante multiplication de la biomasse par quatre par rapport aux plants du type sauvage.
PCT/US2011/026368 2010-02-26 2011-02-25 Plants de riz et de soja transgéniques dotés de caractéristiques de croissance améliorées Ceased WO2011106734A1 (fr)

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US12/660,506 2010-02-26
US12/660,506 US20110030104A1 (en) 2008-08-29 2010-02-26 Nucleic acids encoding plant glutamine phenylpyruvate transaminase (GPT) and uses thereof
US12/660,501 2010-02-26
US12/660,501 US20110030089A1 (en) 2008-08-29 2010-02-26 Transgenic plants with enhanced growth characteristics
US12/660,508 US20110004961A1 (en) 2008-08-29 2010-02-26 Plant glutamine phenylpyruvate transaminase gene and transgenic plants carrying same
US12/660,508 2010-02-26

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DE202012004493U1 (de) 2012-05-05 2012-06-06 Wdt-Wolz-Dental-Technik Gmbh Sintereinsatz für einen Sinterofen zum sauerstofffreien Sintern von Metall oder Keramik
CN107142261A (zh) * 2017-06-02 2017-09-08 武汉生物工程学院 一种在水稻幼穗特异表达的启动子及其应用
US9862964B2 (en) 2008-08-29 2018-01-09 Los Alamos National Security, Llc Transgenic plants with enhanced growth characteristics
CN108056181A (zh) * 2018-01-29 2018-05-22 克东禹王大豆蛋白食品有限公司 一种果蔬千叶豆腐及其制作方法
US10119127B2 (en) 2008-08-29 2018-11-06 Los Alamos National Security, Llc Nucleic acids encoding plant glutamine phenylpyruvate transaminase (GPT) and uses thereof
CN109022483A (zh) * 2018-09-11 2018-12-18 西北农林科技大学 Trv载体介导的病毒诱导基因沉默系统接种牡丹花器官的方法

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US20040123343A1 (en) * 2000-04-19 2004-06-24 La Rosa Thomas J. Rice nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement
WO2006076423A2 (fr) * 2005-01-12 2006-07-20 Monsanto Technology, Llc Genes et leurs utilisations pour ameliorer des plantes
US20090070897A1 (en) * 2006-01-12 2009-03-12 Goldman Barry S Genes and uses for plant improvement
US20100011456A1 (en) * 2005-03-04 2010-01-14 Verenium Corporation Nucleic Acids and Proteins and Methods for Making and Using Them

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US6107547A (en) * 1993-10-06 2000-08-22 New York University Transgenic plants that exhibit enhanced nitrogen assimilation
US20040123343A1 (en) * 2000-04-19 2004-06-24 La Rosa Thomas J. Rice nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement
WO2006076423A2 (fr) * 2005-01-12 2006-07-20 Monsanto Technology, Llc Genes et leurs utilisations pour ameliorer des plantes
US20100011456A1 (en) * 2005-03-04 2010-01-14 Verenium Corporation Nucleic Acids and Proteins and Methods for Making and Using Them
US20090070897A1 (en) * 2006-01-12 2009-03-12 Goldman Barry S Genes and uses for plant improvement

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9862964B2 (en) 2008-08-29 2018-01-09 Los Alamos National Security, Llc Transgenic plants with enhanced growth characteristics
US10119127B2 (en) 2008-08-29 2018-11-06 Los Alamos National Security, Llc Nucleic acids encoding plant glutamine phenylpyruvate transaminase (GPT) and uses thereof
DE202012004493U1 (de) 2012-05-05 2012-06-06 Wdt-Wolz-Dental-Technik Gmbh Sintereinsatz für einen Sinterofen zum sauerstofffreien Sintern von Metall oder Keramik
CN107142261A (zh) * 2017-06-02 2017-09-08 武汉生物工程学院 一种在水稻幼穗特异表达的启动子及其应用
CN107142261B (zh) * 2017-06-02 2020-11-03 武汉生物工程学院 一种在水稻幼穗特异表达的启动子及其应用
CN108056181A (zh) * 2018-01-29 2018-05-22 克东禹王大豆蛋白食品有限公司 一种果蔬千叶豆腐及其制作方法
CN109022483A (zh) * 2018-09-11 2018-12-18 西北农林科技大学 Trv载体介导的病毒诱导基因沉默系统接种牡丹花器官的方法

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