RU2582260C2 - Transgenic plants with enhanced growth characteristics - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry, particularly to a transgenic plant which demonstrates an increase in biomass compared to a wild-type counterpart or untransformed plant comprising a glutamine phenylpyruvate transaminase transgene and a glutamine synthetase transgene, wherein each GPT transgene and GS transgene is operably linked to a plant promoter, as well as a seed to produce said plant. Also disclosed are methods of producing transgenic plants having increased production of 2-oxoglutaramate using glutamine phenylpyruvate transaminase transgene and glutamine synthetase transgene.

EFFECT: invention enables to efficiently produce a transgenic plant with increased biomass.

40 cl, 20 dwg, 16 tbl, 24 ex

Description

The present invention was made with government support under Agreement No. W-7405-ENG-36, which confirms the support provided by the US Department of Energy to the University of California, and according to Agreement No. DE-AC52-06NA25396, which confirms the support provided by the US Department of Energy to Los Alamos National Security, LLC. The government has certain rights to the present invention.

Related Applications

This application claims the priority of provisional application for US patent No. 61/190,520, filed August 29, 2008

State of the art

As the world's population grows and suitable agricultural land continues to be destroyed or otherwise damaged, the need for more efficient and rational agricultural systems is one of the primary problems facing the human race. Increasing the yield, protein content and plant growth rate are the main tasks that must be solved in the process of developing agricultural systems, which can more effectively contribute to solving the problem.

In recent years, the need for improved crop production technologies has only increased, since the yield in the case of many well-developed crops has a tendency to decrease. Many agricultural indicators depend on time, and costs and income depend on the rate of turnover of crops or on the time to market for a product. Therefore, the rapid growth of the plant is an economically important task for many sectors of agricultural activity associated with valuable crops, vegetables, berries and other fruits.

Genetic engineering has played and continues to play an increasingly important, but still controversial, role in the development of sustainable agricultural technologies. A large number of genetically modified plants and related technologies have already been developed in recent years, many of which are now widely used (Factsheet: Genetically Modified Crops in the United States, Pew Initiative on Food and Biotechnology, August 2004, (pewagbiotech .org / resources / factsheets)). The distribution of transgenic plant varieties is very significant today and has increased significantly compared to 2006, when the number of acres of land sown with transgenic plants was about 250 million.

While technologies for producing transgenic plants are gradually spreading, especially in the USA, Canada and Australia, in many areas of the world, especially in Europe, the introduction of genetically modified plants is very slow. Therefore, according to the objectives of the development of rational and efficient agriculture, there is an interest in the development of genetically modified plants, which allow avoiding the problems associated with the introduction of toxins and other potentially dangerous substances into plants and / or the environment. There is also an interest in minimizing the costs of solving the necessary tasks, such as increasing tolerance to herbicides, resistance to pesticides and diseases, and increasing the overall yield. Accordingly, there is a need for transgenic plants that can satisfy these requirements.

The goal of rapid plant growth has been achieved through numerous studies of various growth-regulating systems, many of which are still not fully understood. In particular, the regulatory mechanisms of plants that coordinate the metabolism of carbon and nitrogen are still not fully understood. It is suggested that these regulatory mechanisms have a fundamental effect on plant growth and development.

The metabolism of carbon and nitrogen in photosynthetic organisms should be coordinated to ensure the efficient use of plant resources and energy. Currently, details of some stages of carbon and nitrogen metabolism and metabolic pathways, which are subsystems of larger systems, are known. In photosynthetic organisms, carbon metabolism begins with CO ₂ fixation, which occurs through two large processes called C-3 and C-4 metabolism. In plants with a C-3 metabolism, the enzyme ribulose bisphosphate carboxylase (RuBisCo) catalyzes the combination of CO ₂ with ribulose bisphosphate to produce 3-phosphoglycerate, a tri-carbon compound (C-3), which is used by the plant to synthesize carbon-containing compounds. In plants with C-4 metabolism, CO ₂ combines with phosphoenolpyruvate to form acids containing four carbon (C-4) during the reaction catalyzed by phosphoenolpyruvate carboxylase. Acids are transferred to the cells of the lining of the vascular bundles, where they are decarboxylated with the release of CO ₂ , which then combines with ribulose bisphosphate by the same reaction that is used in C-3 plants.

In the course of numerous studies, it was found that various metabolites are important in the regulation of nitrogen metabolism in plants. These compounds include organic acid malate, as well as the amino acids glutamine and glutamic acid. Nitrogen is assimilated by photosynthetic organisms through the action of the enzyme glutamine synthetase (GS), which catalyzes the association of ammonia with glutamic acid, with the formation of glutamine. GS plays a key role in the assimilation of nitrogen in plants by catalyzing the binding of ammonia to glutamic acid to produce glutamine during the ATP-dependent reaction (Miflin and Habash, 2002, Journal of Experimental Botany, Vol. 53, No. 370, pp.979 -987). GS also reassimilates ammonia released as a result of photo-respiration and breakdown of proteins and nitrogen-bearing compounds. GS enzymes can be divided into two general classes, one of which is represented by the cytoplasmic form (GS1) and the other plastid (i.e., chloroplast) form (GS2).

Previous work has shown that elevated levels of GS1 expression lead to increased levels of GS activity and increased plant growth, although reports are inconsistent. For example, Fuentes et al. reported that CaMV S35 promoter-driven overexpression of Alfalfa GS1 (cytoplasmic form) in tobacco plants resulted in increased levels of GS expression and GS activity in leaf tissue, increased growth during nitrogen starvation, but did not affect growth under optimal nitrogen fertilizer conditions (Fuentes et al., 2001, J. Exp. Botany 52: 1071-81). Temple et al. reported that transgenic tobacco plants overexpressing the full length Alfalfa GS1 coding sequence had significantly elevated levels of GS transcript and GS polypeptide, which combined into an active enzyme, but no phenotypic effects on growth were reported (Temple et al., 1993, Molecular and General Genetics 236: 315-325). Corruzi et al. reported that transgenic tobacco plants overexpressing the transgene of the cytosolic GS1 pea under the control of the CaMV S35 promoter showed increased GS activity, increased levels of the cytosolic GS protein and improved growth characteristics (US Patent No. 6,107,547). Unkefer et al. More recently, it has been reported that transgenic tobacco plants over-expressing Alfalfa GS1 in leaf tissues, which were examined for increased leaf-to-root GS activity after self-pollination, achieving an increased copy number of the GS1 transgene, were found to produce increased levels of 2-hydroxy-5-oxoproline in their leaf parts, which was found to lead to markedly increased growth rates compared to wild-type tobacco plants (see US Patents Nos. 6,555,500, 6,593,275 and 6,831,040).

Unkefer et al. found that the use of 2-hydroxy-5-oxoproline (also known as 2-oxoglutaramate) improves plant growth (US Patent Nos. 6,555,500; 6,593,275; 6,831,040). In particular, Unkefer et al. found that elevated concentrations of 2-hydroxy-5-oxoproline in leaf tissues (relative to root tissues) trigger a cascade of events that lead to improved plant growth characteristics. Unkefer et al. described methods by which the concentration of 2-hydroxy-5-oxoproline in leaf parts can be increased in order to trigger events leading to improved plant growth characteristics, especially by applying a solution of 2-hydroxy-5-oxoproline directly to leaf parts of a plant and overexpression of glutamine synthetase directly in leaf tissues.

Transaminase and hydrolyase enzymes known to be involved in the synthesis of 2-hydroxy-5-oxoproline in animals have been found in animal and liver tissues of animals (Cooper and Meister, 1977, CRC Critical Reviews in Biochemistry, pages 281-303; Meister, 1952, J. Biochem. 197: 304). In plants, the biochemical synthesis of 2-hydroxy-5-oxoproline is already known, but poorly described. Moreover, the function of 2-hydroxy-5-oxoproline in plants and the value of the size of its deposits (concentration in tissue) are unknown. Finally, no specific data can be obtained from the prior art that transaminase (s) or hydrolase (s) can exist and / or be active in catalysing the synthesis of 2-hydroxy-5-oxoproline in plants, and it cannot be obtained from the prior art information about any isolated or described plant transaminases.

SUMMARY OF THE INVENTION

The present invention relates to transgenic plants showing significantly increased growth rates, a higher yield of seeds and fruits / pods, earlier or more productive flowering, more efficient utilization of nitrogen, increased tolerance to high salt conditions and increased biomass yield. In one embodiment of the present invention, transgenic plants are modified in such a way that overexpression of both glutamine-phenylpyruvate transaminase (GPT) and glutamine synthetase (GS) is provided. GPT + GS double transgenic plants of the present invention exhibit improved growth characteristics, wherein the TO generation lines show an increase in biomass compared to wild-type counterparts from 50% to 300%. Generations obtained by sexual crossing and / or self-pollination, as a rule, have even better characteristics, with some double-transgenic plants achieving an amazing four-fold increase in biomass compared to wild-type plants. Similarly, the yield of flowers and fruits or pods is also significantly improved, with the generation TO lines, as a rule, showing an increase of 50% -70%, compared with wild-type analogues, and in some cases an increase of 100% is achieved. Transgenic plants exhibiting such improved phenotypic growth characteristics have been successfully generated across a spectrum of individual plant species using various transformation techniques, various expression vectors and promoters, and heterologous and homologous transgenic sequences from various species, numerous examples are provided herein. The present invention, therefore, reveals a fundamental revolutionary technique by which it is possible to exert a positive influence on the development of almost all areas of agriculture.

Applicants have discovered that the enzyme glutamine-phenylpyruvate transaminase (GPT) is a catalyst for the synthesis of 2-hydroxy-6-oxoproline (2-oxoglutaramate) in plants. 2-Oxoglutaramate is a powerful signal metabolite that regulates the functions of a large number of genes involved in the photosynthesis apparatus involved in carbon fixation and nitrogen metabolism. The present invention discloses isolated nucleic acid molecules encoding GPT, and for the first time claims that the encoded enzyme is directly involved in the synthesis of 2-hydroxy-5-oxoproline. This object of the present invention is described through the disclosure of GPT polynucleotides encoding several types of GPTs, including Arabidopsis, Grapes, Rice, Soy, Barley, and non-plant homologue from Striped Percina, most of which are expressed as recombinant GPTs and are confirmed to have GPT activity .

The present invention also discloses transgenic plants that express both the GPT transgene and the GS transgene. The expression of these two transgenes in such “double-transgenic” plants leads to a significantly increased rate of carbon dioxide fixation and an extremely enhanced growth effect, since these plants show very significant and even staggeringly increased growth rates and yields of flowers / fruits / pods / seeds. Also provided are methods for producing such transgenic plants with enhanced growth.

By preferably increasing the concentration of the signal metabolite of 2-oxoglutaramate (i.e., in leaf tissues), the transgenic plants of the present invention are able to produce higher yields in shorter periods of time, and therefore it is possible to provide increased productivity in the agricultural industry for a wide variety of crops. It is important to note that, unlike the numerous transgenic plants described to date, according to the present invention, natural plant genes encoding a natural plant enzyme are used. The improved growth characteristics of the transgenic plants of the present invention are achieved, essentially, by introducing additional GPT and GS enzymatic ability in plants. Thus, the transgenic plants of the present invention do not express any toxic substances, growth hormones, products of viral or bacterial genes and, thus, are free from much of what previously prevented the approval of transgenic plants in some parts of the world.

In one embodiment of the present invention, the present invention provides a transgenic plant comprising a GPT transgene and a GS transgene, wherein said GPT transgene and said GS transgene is operably linked to a plant promoter. In a specific embodiment, the GS transgene is a GS1 transgene. In another specific embodiment of the present invention, 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 and SEQ ID NO: 36, and (b) an amino acid sequence that is at least 75% identical to the amino acid sequence 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 and SEQ ID NO: 36 and has GPT activity. In another specific embodiment of the present invention, the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 4 and SEQ ID NO: 7 from residue 11, and (b) an amino acid sequence that at least 75% identical to SEQ ID NO: 4 or SEQ ID NO: 7. In some embodiments of the present invention, GPT and GS transgenes are included in the plant genome. The transgenic plant of the present invention may be a monocotyledonous or dicotyledonous plant.

The present invention also claims the offspring of any generation of transgenic plants of the present invention, wherein said offspring contains a GPT transgene and a GS transgene, as well as the seeds of any generation of transgenic plants of the present invention, where said seeds comprise said GPT transgene and said GS transgene. The transgenic plants of the present invention may exhibit one or more improved growth rate characteristics compared to wild-type counterparts or untransformed plants, including, but not limited to, increased growth rate, increased biomass yield, increased seed yield, flower or bud crop, harvest of fruits or pods, larger leaves, and many also exhibit elevated levels of GPT and / or GS activity, and / or elevated levels of 2-oxoglutaramate. In some embodiments of the present invention, transgenic plants of the present invention show increased nitrogen utilization efficiency or increased tolerance to saline soil or salt conditions.

Methods for producing the transgenic plants of the present invention and their seeds are also disclosed in the present invention, including methods for producing plants having enhanced growth properties, increased nitrogen utilization efficiency, and increased tolerance to germination or growth in saline soil or salt conditions, relative to wild-type or untransformed analogues plants.

Brief Description of the Drawings

The materials of the patent or patent application contain at least one drawing made in color. Copies of a patent or publication of a patent application with a color drawing (s) will be provided if the required fee is paid and paid.

In FIG. 1. nitrogen assimilation and biosynthesis of 2-oxoglutaramate is shown, the metabolic pathway is shown.

In FIG. 2. a photograph showing a comparison of transgenic tobacco plants overexpressing either GS1 or GPT compared to a wild-type tobacco plant. From left to right: wild-type plant, Alfalfa GS1 transgene, Arabidopsis GPT transgene. See examples 3 and 5 below.

In FIG. 3. a photograph showing a comparison of transgenic Micro-Tom tomato plants overexpressing either GS1 or GPT compared to a wild-type tomato plant. From left to right: wild-type plant, Alfalfa GS1 transgene, Arabidopsis GPT transgene. See examples 4 and 6 below.

In FIG. 4. photos are shown showing a comparison of leaf sizes for wild-type and GS1 or GPT transgenic tobacco plants. A: Comparison of GS1 leaves of transgenic tobacco (basal leaf) and wild type (top leaf). B: Comparison of the leaves of GPT of transgenic tobacco (basal leaf) and wild type (top leaf).

In FIG. 5. photos are shown showing a comparison of transgenic tobacco plants obtained as a result of various crosses between GS1 and GPT transgenic tobacco lines, with wild type and transgenic plants once. A-C: Cross 2, 3 and 7, respectively. See example 7 below.

In FIG.6. photos are shown showing a comparison of leaf sizes of wild-type plants and crosses between GS1 and GPT transgenic tobacco plants. A: Comparison of the leaves of GSXGPT Cross 3 (basal leaf) and wild type (upper leaf). B: Comparison of GSXGPT Cross 7 leaves (basal leaf) and wild type (upper leaf). See example 7 below.

FIG. 7. a photograph of a transgenic pepper plant (right) and a control wild-type pepper plant (left) is shown, showing a higher yield of pepper fruits in the case of a transgenic plant compared to a wild-type control plant. See example 8 below.

In FIG. 8. a comparison of transgenic bean plants (several transgenic lines expressing Arabidopsis GPT and GS transgenes) and wild-type control bean plants is given. Above left: plant height on various days; Above right: number of buds; Bottom left: number of flowers; Bottom right: number of bean pods. The wild type is a control, and 2A, 4A and 5B are lines of transgenic plants. See example 9 below.

FIG. 9. A photograph of a transgenic bean plant (right) and a control wild-type bean plant (left) showing enhanced growth of a transgenic plant relative to a wild-type control plant is shown. Transgenic line expressing Arabidopsis GPT and GS transgenes. See example 9 below.

FIG. 10. Buds, flowers, and pods of transgenic bean plants compared to control wild-type bean plants (transgenic line expressing GPT and Arabidopsis GS grape transgenes). See example 10 below.

FIG. 11. Photograph of a transgenic bean plant (right) and a control wild-type bean plant (left) showing enhanced growth of a transgenic plant compared to a wild-type control plant. The transgenic line expresses transgenes of grapes GPT and Arabidopsis GS. See example 10 below.

FIG. 12. Transgenic plants of Vigna line A are shown in comparison with control plants of wild type Vigna (transgenic line expressing Arabidopsis GPT and GS transgenes), it can be seen that transgenic plants grow faster, and flowers and pods form faster compared to control plants of wild type. (A) Measurements of the relative height and length of leaves on May 21, (B) The relative number of three-leafed leaves and buds on June 28, (C) The relative number of flowers, buds and pods on June 22. See example 11 below.

FIG.13. Photo of a transgenic plant of Vigna line A (right) and a control plant of wild-type Vigna (left) showing increased growth of a transgenic plant compared to a control plant of wild type. Transgenic line expressing Arabidopsis GPT and GS transgenes. See example 11 below.

FIG. 14. Transgenic plants G-line vignas compared to control plants Wild-type vignas (a transgenic line expressing the GPT transgene from grapes and Arabidopsis GS transgene), it can be seen that transgenic plants grow faster and flowers and pods are formed earlier compared to wild-type control plants . (A) the height of plants, (B) the number of flowers and pods (C) the number of leaf buds and three-leafed leaves. See example 12 below.

FIG. 15. Photo of the transgenic plant Vigna G line (right) and the control plant Wild type Vigna (left), as you can see, transgenic plants have increased growth compared to wild type plants. The transgenic line expresses the GPT transgene from grapes and the Arabidopsis GS transgene. See example 12 below.

FIG.16. Photo of a transgenic Cantaloupe plant (right) and a wild-type Cantaloupe control plant (left) showing increased growth of a transgenic plant relative to a wild-type control plant. The transgenic line expresses Arabidopsis GPT and GS transgenes. See example 14 below.

FIG. 17. Photo of transgenic common pumpkin plants (right) and control wild-type pumpkin plants (left), showing increased growth of transgenic plants relative to wild-type control plants. The transgenic line expresses Arabidopsis GPT and GS transgenes. See example 15 below.

FIG. 18. Photograph of Arabidopsis transgenic plants (right) and wild type Arabidopsis control plants (left) showing increased growth of transgenic plants compared to wild type control plants. The transgenic line expresses Arabidopsis GPT and GS transgenes. See example 16 below.

FIG. 19. Transgenic tomato plants expressing Arabidopsis GPT and GS transgenes compared to control wild-type tomato plants. (A) A photograph of the leaves of a transgenic tomato plant (right) compared to the leaves of a wild-type control plant (left), showing larger leaves in the case of a transgenic plant. (B) Photograph of transgenic tomato plants (right) and wild-type control plants (left) showing increased growth of transgenic plants compared to wild-type control plants. See example 17 below.

FIG 20. A photograph of the transgenic Camelina plant (right) and the wild type Camelina control plant (left) showing increased growth of the transgenic plant relative to the wild type control plant are shown. The transgenic line expresses Arabidopsis GPT and GS transgenes. See example 18 below.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

Unless otherwise indicated, all terms known from the technical field, naming systems and other scientific terminology used in this document are intended to have meanings generally understood by those skilled in the art to which the present invention relates. In some cases, terms with understandable, in general, meanings are defined herein for clarity and / or for convenient reference, and the inclusion of such definitions in this document should not be construed as a significant difference between these terms from terms that are generally known from the field technicians. The techniques and procedures described herein or referenced herein are generally well understood and commonly used by those skilled in the art using conventional methods, such as, for example, the widely used molecular cloning methods described by Sambrook et al ., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (Ausbel et al., Eds., John Wiley & Sons, Inc. 2001; Transgenic Plants: Methods and Protocols (Leandro Pena, ed., Humana Press, 1 ^st edition, 2004); and, Agrobacterium Protocols (Wan, ed., Humana Press, 2 ^nd edition, 2006.) Accordingly, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and / or parameters specified by the manufacturer, unless otherwise specified.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and their polymers ("polynucleotides") either in single or double stranded form. Unless specifically limited, the term "polynucleotide" encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as said nucleic acid and are metabolized in a similar manner to nucleotides of natural origin. Unless otherwise indicated, a specific nucleic acid sequence also implies in an implied manner its conservatively modified variants (for example, substitutions associated with the degeneracy of the genetic code) and complementary sequences, as well as the sequence indicated unambiguously. Thus, substitutions associated with the degeneracy of the genetic code can be made by obtaining sequences in which the third position of one or more (or all) of the codons is replaced by a 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). The term nucleic acid is used interchangeably with the terms gene, cDNA, and mRNA encoded by a gene.

The term "promoter" refers to regulatory nucleic acid sequences or sequences that direct the transcription of operably linked nucleic acids. As used herein, the term “plant promoter” is a promoter that functions in plants. Promoters include the necessary nucleic acid sequences near the site of transcription initiation, such as, for example, in the case of a type II polymerase promoter, a TATA element. The promoter also optionally includes elements such as a distal enhancer or repressor, which can be located up to several thousand base pairs from the transcription site. A "constitutive" promoter is a promoter that is active under most environmental and experimental conditions. An “inducible” promoter is a promoter that is active in regulating environmental and experimental conditions. The term "operably linked" refers to a functional relationship between a sequence that regulates the expression of a nucleic acid (such as a promoter, or an array of transcription factor binding sites), and a second nucleic acid sequence, where the expression control sequence directs the transcription of the nucleic acid in a second sequence.

The terms "polypeptide", "peptide" and "protein" in the context of the present description are used interchangeably to refer to a polymer consisting of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemically obtained mimetic of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as amino acids that are subsequently modified, for example, hydroxyproline, gamma-carboxyglutamine and O-phosphoserine. The term "amino acid analogues" refers to compounds that have the same basic chemical structure as the naturally occurring amino acid, i.e. have a carbon atom in the alpha position, which is bonded to hydrogen, a carboxyl group, an amino group and an R group, for example homoserine, norleucine, methionine sulfoxide and methionine methyl sulfonium. Such analogues carry modified R-groups (for example, norleucine) or a modified peptide framework, but retain the same chemical structure as the naturally occurring amino acid. The term "amino acid mimetics" refers to chemical compounds that have a structure different from the general chemical structure of an amino acid, but which function similarly to the naturally occurring amino acid.

Amino acids can be referred to in this document either through well-known three-letter characters, or by single-letter characters recommended by the IUPAC-IIB Commission on Biochemical Nomenclature. Nucleotides, in a similar manner, may be referred to by well-known single-letter codes.

The term "plant" includes the whole plant, plant organs (for example, leaves, stems, flowers, roots, reproductive organs, embryos and parts thereof, etc.), seedlings, seeds, plant cells and its offspring. The class of plants that can be used in the method of the present invention, in General, is a class of higher plants, amenable to the method of transformation, including hidden seed plants (monocotyledonous and dicotyledonous plants), as well as gymnosperms. These include plants of various levels of ploidy, including polyploids, diploids, haploids and hemizygous.

The terms “GPT polynucleotide” and “GPT nucleic acid” are used interchangeably herein and refer to a polynucleotide sequence, full or partial length, of a gene that encodes a polypeptide involved in the catalysis of the synthesis of 2-oxoglutaramate and includes polynucleotides containing as translated ( coding) and untranslated sequences, as well as their complements. The term "GPT coding sequence" refers to a portion of a gene that is transcribed and encodes a GPT protein. The term “target sequence” refers to the amino terminal portion of a protein that directs the protein to the subcellular compartment of a cell, such as chloroplast in a plant cell. GPT polynucleotides are further determined by their ability to hybridize when determined with the GPT polynucleotides disclosed herein, or with PCR products derived from them.

A "GPT transgene" is a nucleic acid molecule containing a GPT polynucleotide that is exogenous to a transgenic plant or embryo, organ or seed of a plant containing a nucleic acid molecule, or which is exogenous to an ancestor of a transgenic plant containing a GPT polynucleotide, its germ, organ or seed.

The terms “GS polynucleotide” and “GS nucleic acid” are used interchangeably herein and refer to a polynucleotide sequence, full or partial length, of a gene that encodes a protein for glutamine synthetase and includes polynucleotides containing both translated (coding) and untranslated sequences, as well as their complements. The term "GS coding sequence" refers to the part of a gene that is transcribed and encodes a GS protein. The terms “GS1 polynucleotide” and “GS1 nucleic acid” are used interchangeably herein and refer to a polynucleotide sequence, full or partial length, of a gene that encodes a glutamine synthetase protein of isoform 1 and includes polynucleotides containing both translated (encoding) and untranslated sequences, as well as their complements. The term "GS1 coding sequence" refers to the part of a gene that is transcribed and encodes a GS1 protein.

A “GS transgene” is a nucleic acid molecule containing a GS polynucleotide that is exogenous to a transgenic plant, or the germ, organ or seed of a plant containing a nucleic acid molecule, or which is exogenous to an ancestor of a transgenic plant containing a GS polynucleotide, its germ, organ or seed. A "GS1 transgene" is a nucleic acid molecule containing a GS1 polynucleotide that is exogenous to a transgenic plant, or the germ, organ or seed of a plant containing a nucleic acid molecule, or which is exogenous to an ancestor of a transgenic plant containing a GS1 polynucleotide, its germ, organ or seed.

Examples of GPT polynucleotides of the present invention are provided herein and include the GPT coding sequence for GPTs of Arabidopsis, rice, barley, bamboo, soy, grapes and striped pepper.

Partial length GPT polynucleotides include polynucleotide sequences encoding the truncated at the N- or C-terminus of the GPT, mature GPTs (without a target sequence), as well as sequences encoding the GPT domains. Examples of GPT polynucleotides encoding truncated at the N-terminus of GPT include -30, -45 and -56 Arabidopsis constructs, in which the coding sequences for the first 30, 45 and 56 amino acids are excluded, respectively, in the full length GPT structure according to SEQ ID NO: 2 .

When using the GPT polynucleotides of the present invention to produce transformed cells and transgenic plants, one skilled in the art can determine that the inserted polynucleotide sequence may not be identical, but only “substantially identical” to the sequence of the gene from which it originated, as defined below. The term "GPT polynucleotide" particularly encompasses such substantially identical variants. Similarly, one of skill in the art can determine that, as a result of the degeneracy of the genetic code, a series of polynucleotide sequences will encode the same polypeptide, and this means that all such polynucleotide sequences are included within the scope of the term GPT polynucleotide. In addition, this term, in particular, includes sequences (as defined below) that are substantially identical to the GPT of the polynucleotide sequence disclosed herein and those that encode polypeptides that are either wild type GPT mutants of the GPT or that retain the GPT function of the polypeptide (for example, result from conservative substitutions of amino acids in the GPT polypeptide). The term "GPT polynucleotide", therefore, also includes such essentially identical variants.

The term “conservatively modified variants” applies to both amino acid sequences and nucleic acid sequences. In relation to specific nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or, in the case where the nucleic acid does not encode an amino acid sequence, to substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where alanine is defined by a codon, this codon can be changed to any of the corresponding codons without changing the encoded polypeptide. Such “silent variation” nucleic acid variations are one type of conservatively modified variation. Each nucleic acid sequence of the present invention that encodes a polypeptide also describes each possible silent variation of the nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (with the exception of AUG, which is a codon only for methionine, and TGG, which is a codon only for tryptophan) can be modified to produce a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implied in each described sequence.

With respect to amino acid sequences, one of ordinary skill in the art knows that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that result in a change, deletion, or addition of a single amino acid or a small fraction of the amino acids in the encoded sequence are "conservatively modified variant", where the change leads to the replacement of amino acids with chemically similar amino acids. Conservative substitution tables showing functionally similar amino acids are well known in the art. Such conservatively modified variants, in addition, do not exclude polymorphic variants, interspecific homologs and alleles of the invention.

Each of the following 8 groups contains amino acids, which are conservative substitutions for each other:

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, for example, Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can be defined in terms of various levels of organization. For a general review of this organization, see, e.g., Alberts et al, Molecular Biology of the Cell ( 3 ^rd ed, 1994.) And Cantor and Schimmel, Biophysical Chemistry Part I:. The Conformation of Biological Macromolecules ( 1980). The term "primary structure" refers to the amino acid sequence of a particular peptide. The term "secondary structure" refers to locally located, three-dimensional structures within the polypeptide. These structures are generally known as domains. Domains are portions of a polypeptide that form a compact unit of a polypeptide and are typically in length from about 25 to about 500 amino acids. Typical domains consist of sections of lower organization, such as sections of the β-layer and α-helix. The term "tertiary structure" refers to the completed three-dimensional structure of the polypeptide monomer. The term “quaternary structure” refers to a three-dimensional structure formed by a non-covalent compound of independent tertiary elements. Anisotropic constituents are also known as energy constituents.

The term "isolated" refers to a substance that is mainly or substantially combined from components that, as a rule, accompany the substance in the form in which it is found in its native or natural state. However, the term "isolated" is not intended to refer to components present in an electrophoretic gel or other separation medium. The isolated component is free from such a separation medium and is in a form ready for use in another application or already in use in a new / new application / environment. An “isolated” antibody is an antibody that has been identified and isolated and / or recovered from its environmental components in vivo. The polluting components of its natural environment are substances that will interfere with the diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and protein or non-protein dissolved substances. In preferred embodiments of the present invention, the antibody is purified (1) by more than 95% by weight of the antibody, as determined using the Lowry method, and most preferably by more than 99% by weight (2) to a degree sufficient to produce at least 15 residues of the N-terminal or internal amino acid sequence by using a rotary beaker sequencer, or (3) until homogeneous by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) in reducing or non-reducing In conditions under pressure, using Coomassie blue or, preferably, silverfish. Isolated antibodies include an in situ antibody within recombinant cells since at least one component of the antibody’s natural environment will not be present. Typically, however, an isolated antibody is obtained by at least one purification step.

The term “heterologous” when applied to parts of a nucleic acid indicates that the nucleic acid contains two or more sequences that are found to be in nature not equally related to each other. For example, a nucleic acid is typically produced recombinantly, having two or more sequences of unrelated genes arranged to create a new functional nucleic acid, for example, a nucleic acid that encodes a protein from one source and encodes a peptide sequence from another source. Similarly, a heterologous protein indicates that the protein contains two or more sequences that are not found to be in the same degree of kinship with each other (for example, a hybrid protein).

The terms “identical” or “percent identity”, in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a certain percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity for a specific area, when comparing and aligning for maximum match in the comparison window or area, as measured using sequence comparison algorithms, or by manual alignment and visual inspection.This definition also applies to the complement of a test sequence that has significant complementarity to sequences or subsequences when the test sequence is substantially identical to the control sequence. to the complement of the test sequence, which has a significant complementary sequence of sequences or subsequences when the test sequence is substantially identical to the control sequence.

When a percent sequence identity is applied to polypeptides, it was found that the positions of residues that are not identical often differ due to conservative amino acid substitution, when amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), and therefore functional properties the polypeptide does not change. When sequences differ due to conservative substitutions, the percentage of sequence identity can be increased in order to correct for the conservative nature of substitution.

To compare sequences, as a rule, one sequence performs the functions of a control sequence with which the test sequence is compared. When applying the sequence comparison algorithm, the test and control sequences are entered into the computer, if necessary, the coordinates of the sequences are indicated, and the program parameters of the sequence comparison algorithm are indicated. The default program parameters may be applied, or alternative parameters may be indicated. The sequence comparison algorithm then calculates the percentage of identity of the test sequence with respect to the control sequence based on the program parameters.

In the context of the present description, the term "comparison window" refers to a fragment containing any number of consecutive positions consecutively selected from the group consisting of from 20 to 600, typically from about 50 to about 200, more often from about 100 to about 150 positions in which the sequence can be compared with a control sequence with the same number of adjacent positions after the optimal alignment of the two sequences. Sequence alignment methods for comparison are well known in the art. Optimal sequence alignment for comparison can be carried out, for example, using the Smith-Waterman local homology algorithm, 1981, Adv. Appl. Math. 2: 482, by the Niedemann-Wunsch homology matching algorithm, 1970, J. Mol. Biol. 48: 443, by the method of searching for Pearson-Lipman affinities, 1988, Proc. Nat'l. Acad. Sci. USA 85: 2444, through computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics, Genetics Computer Group, 575 Science Dr. Dr. Madison, WI), or by manual alignment and visual inspection (see e.g. Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

Preferred examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nuc. Acids Res. 25: 3389-3402 and Altschul et al., 1990, J. Mol. Biol. 215: 403-410, respectively. BLAST and BLAST 2.0 are typically used with the default parameters described in this document to determine the percent sequence identity for the nucleic acids and proteins of the present invention. BLAST analysis software is publicly available through the National Center for Biotechnology Information (NCBI). This algorithm first involves identifying high-score pairs of sequences (HSPs) by identifying short words of length W in the requested sequence that either match or satisfy some positively evaluated threshold score T when matching with a word of the same length in the database sequence. T is denoted as the ballroom threshold of adjacent words (Altschul et al., Above). These initial hits (matches) of neighboring words act as starting points for finding longer HSPs containing them. Word hits are distributed in both directions along each of the two compared sequences until the cumulative matching score can increase. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (incentive score for a pair of matching residues; always> 0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a weight matrix is used to calculate the cumulative score. The distribution of word hits is stopped when: the cumulative comparison score falls X times from the maximum value achieved; cumulative score goes to zero or lower; or the end of any sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of matching. The BLAST program (for nucleotide sequences) uses the default word length (W) 11, expectation (E) 10, M = 5, N = -4 and compare both chains. For amino acid sequences, the BLAST program uses the default word length (W) 3 and expectation (E) 10, BLOSUM62 score matrix comparisons (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) ( B) 50, expectation (E) 10, M = 5, N = -4 and comparison of both chains.

The BLAST algorithm then performs a statistical analysis of the similarity between the two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the least probability of the sum (P (N)), which provides an indication of the probability with which a coincidence between two nucleotide or amino acid sequences will occur randomly. For example, an amino acid sequence is considered to be similar to a protein, such as a protease, if the least likelihood of a sum compared to the tested amino acid sequence with a protein, such as a protease amino acid sequence, is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The phrase “stringent hybridization conditions” refers to the conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but not to other sequences. Severe conditions are sequence dependent and vary in different environments. Longer sequences, in particular, hybridize at higher temperatures. Extensive guidance on nucleic acid hybridization can be 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). In general, very stringent conditions are chosen about 5-10 ° C below the melting point (Tm) of a particular sequence at a specific ionic pH. Low stringency conditions are generally selected about 15-30 ° C. below Tm. Tm is the temperature (at a certain ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium (since the target sequences are present in excess, at Tm, 50% of the probes acts at equilibrium). Severe conditions are those in which the salt concentration is less than about 1.0 M sodium ions, as a rule, the concentration is from about 0.01 to 1.0 M sodium ions (or other salts) at pH 7.0-8.3 and a temperature of at least about 30 ° C for short probes (e.g., 10-50 nucleotides) and at least about 60 ° C for long probes (e.g., more than 50 nucleotides). Severe conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, the positive signal is at least two-fold background hybridization, preferably ten-fold background hybridization.

Nucleic acids that do not hybridize to each other under stringent conditions, however, are substantially identical if the polypeptides they encode are substantially identical. This happens, for example, when a nucleic acid copy is created using the maximum codon degeneracy allowed by the genetic code. In such cases, nucleic acids typically hybridize under hybridization conditions of moderate stringency.

Genomic DNA or cDNA containing GPT polynucleotides can be identified in standard southern blots under stringent conditions using the GPT polynucleotide sequences disclosed herein. To this end, suitable stringent conditions for such hybridizations are those that include hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37 ° C, and at least one wash in 0.2 X SSC at a temperature at least about 50 ° C, typically from about 55 ° C to about 60 ° C, for 20 minutes, or under equivalent conditions. Positive hybridization is at least twice as large as the background. Those skilled in the art will readily determine that alternative hybridization and washing conditions can be used to provide conditions of similar stringency.

The following indication that the two polynucleotides are essentially identical is that the control sequence amplified by a pair of oligonucleotide primers can then be used as a probe under stringent conditions to isolate a test sequence from a cDNA or genomic library, or to identify a test sequence in for example, a northern or southern blot.

Transgenic plants:

The present invention discloses new transgenic plants exhibiting substantially improved agronomic characteristics, including faster growth, higher raw tissue mass of mature plants and higher total biomass, earlier and more abundant flowering, and a larger yield of fruits, pods and seeds. The transgenic plants of the present invention are obtained by introducing into the plant one or more expressed genetic constructs capable of controlling the expression of one or more polynucleotides encoding glutamine synthetase (C3) and glutamine phenyl pyruvate transaminase (GPT). In an exemplary embodiment of the present invention, parental lines with one transgene are obtained that carry the coding sequence of either the GPT or GS1 transgene, they are preferably self-pollinated until they become homozygous for the transgene, and then are crossed to produce progeny plants containing both transgenes.

In suitable transformation variants of the present invention, one or more copies of the expressed genetic construct are inserted into the genome of the host plant, thereby providing enhanced GS and GPT enzymatic ability in the plant, resulting in increased synthesis of 2-oxoglutaramate, which in turn transmits signal for the expression of the metabolic gene, leading to enhanced plant growth and improvement of other agronomic characteristics. 2-oxoglutaramate is a metabolite that is an extremely potent effector of gene expression, metabolism and plant growth (US Pat. No. 6,555,500), and which can play a central role in coordinating carbon and nitrogen metabolism systems (Lancien et al., 2000, Enzyme Redundancy and the Importance of 2-Oxoglutarate in Higher Plants Ammonium Assimilation, Plant Physiol. 123: 817-824). See also the schematic 2-oxoglutaramate pathway shown in FIG. 1.

As one object of the present invention, the inventors isolated a nucleic acid molecule encoding the Arabidopsis glutamine-phenylpyruvate-transaminase (GPT) enzyme (see Example 1 below), and for the first time demonstrated that the expressed recombinant enzyme is active and is capable of catalyzing the synthesis of the signal metabolite of 2-oxoglutaramate (see Example 2 below). Further, the inventors first demonstrated that overexpression of the Arabidopsis glutamine transaminase gene in a transformed heterologous plant leads to an increase in CO ₂ fixation rates and improved growth characteristics (see Example 3 below).

Applicants' previous work has demonstrated that overexpression of the Alfalfa GS1 gene under the control of a strong constitutive promoter leads to transgenic tobacco plants with higher levels of GS activity in the leaves. These plants are ahead of their wild-type counterparts in growth rate, they fix CO ₂ more quickly, contain increased concentrations of total protein, as well as increased concentrations of glutamine and 2-oxoglutaramate, and show increased rates of nitrate uptake through their roots.

As described herein (see Example 3 below), overexpression of a transgene containing Arabidopsis full length GPT coding sequence in transgenic tobacco plants also leads to faster fixation of CO ₂ and increased levels of total protein, glutamine and 2-oxoglutaramate. These transgenic plants also grow faster than wild-type plants (FIG. 2). Similarly, in previous studies with a tomato plant (see Example 4 below), tomato plants transformed with the Arabidopsis GPT transgene showed significantly increased growth rate, improved flowering, and seed yield compared to wild-type control plants (see FIG. 3 and Example 4 below).

In one specific embodiment of the present invention, using Examples 3, 5 and 7 below, it is shown that the first set of parental tobacco plant lines with one transgene carrying the Alfalfa GS1 gene, including 5 ′ and 3 ′ untranslated regions, were obtained from the use of Agrobacterium-mediated gene transformation, at selective pressure, together with screening for the phenotype with the fastest growth, and self-pollination to homozygosity of the transgene / phenotype (see Example 5 below). A second set of parent lines of a single transgene tobacco plant bearing the full length Arabidopsis coding sequence GPT was obtained in the same way (see Example 3 below). High growth plants selected for each of the parental lines were then sexually crossed to produce offspring lines (see Example 7 below).

The offspring obtained from the numerous crosses of Arabidopsis GS1 and GPT of transgenic tobacco plants shows much better and surprisingly high growth rates compared to parent lines with a single transgene, as well as compared to wild-type plants. FIG. 5 shows photographs of offspring with two transgenes obtained by GS1 X GPT crosses of plants with one transgene, compared with wild-type plants and parent plants with one transgene. FIG.6 shows photographs that allow you to compare the size of the leaves of the offspring with two transgenes and wild-type plants. During the experiments, it was observed that the growth rates of these transgenic plants with two transgenes are 200% -300% higher compared to wild-type plants (see Example 7 below). Moreover, the levels of total biomass are significantly higher for plants with two transgenes, and the raw tissue mass of the whole plant is two to three times the mass of the raw tissue of the wild-type plant. Similarly, seed yields in transgenic plants with two transgenes increase, and two to three times as many seeds are formed compared to wild-type plants, and the total seed yields exceed the yields for wild-type plants by 300-400%.

In addition to the transgenic tobacco plants mentioned above, examples of various other types of transgenic plants containing GPT and GS transgenes are provided herein. As shown in this document, transgenic plants with improved growth characteristics were obtained for two types of Tomato (see Examples 4 and 17), Pepper (Example 8), Beans (Examples 9 and 10), Vigna (Examples 11 and 12), Alfalfa (Example 13), Cantaloupes (Example 14), Pumpkins (Example 15), Arabidopsis (Example 16) and Camilena (Example 18). These transgenic plants of the present invention were obtained using a wide variety of techniques using Agrobacterium-mediated calluses, floral dip transformation, seed inoculation, pod inoculation, and direct flower inoculation, as well as their combinations, and sexual crosses of plants with one transgene , examples of which are given in this document. Various GPT and GS transgenes have been successfully used in the preparation of the transgenic plants of the present invention, as shown herein.

The present invention also provides methods for producing a transgenic plant having enhanced growth and other improved agronomic characteristics. In one embodiment of the present invention, a method for producing a transgenic plant having enhanced growth and other improved agronomic characteristics comprises introducing into the plant cell an expression cassette containing a nucleic acid molecule encoding a GPT transgene under the control of a suitable promoter capable of controlling transgene expression to produce transformed plant cells, and obtaining a transgenic plant that expresses encoded GPT. In another embodiment of the present invention, a method for producing a transgenic plant having enhanced growth and other improved agronomic characteristics comprises introducing into the plant cell one or more nucleic acid constructs or expression cassettes containing a nucleic acid molecule encoding a GPT transgene and a GS transgene, under the control of one or more suitable promoters (and, optionally, other regulatory elements) capable of controlling transgene expression, thereby obtaining trans formed plant cells, and obtaining a transgenic plant that expresses GPT and GS transgenes.

Based on the results set forth herein, it is clear that any number of GPT and GS polynucleotides can be used to produce the transgenic plants of the present invention. Both GS1 and GPT proteins are very conserved among various plant species, and from the experimental data presented in this document, it is obvious that closely related non-plant GPTs can also be used well (for example, Danio rerio GPT). With respect to GPT, many GPT polynucleotides derived from various species have been shown to be active and useful as GPT transgenes. Similarly, various GS polynucleotides can be used, including, but not limited to, any plant GS1 encoding a polynucleotide that generates GS activity in a host cell transformed with an expressed GS1 construct.

In a specific embodiment of the present invention, the GPT transgene is a GPT polynucleotide encoding a GPT derived from Arabidopsis, such as GPT sequences of SEQ ID NO: 2, SEQ ID NO: 21 and SEQ ID NO: 30, and the GS transgene is a GS polynucleotide coding for originating from Alfalfa GS1 (i.e., SEQ ID NO: 4) or originating from Arabidopsis GS1 (SEQ ID NO: 7). The GPT transgene can be encoded by the nucleotide sequence of SEQ ID NO: 1; a nucleotide sequence of at least 75%, and more preferably at least 80%, identical to SEQ ID NO: 1 and encoding a polypeptide having GPT activity; a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2, or a polypeptide having at least 75% and more preferably at least 80% identical sequence and having GPT activity; and a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2 truncated at its amino ends by 30 to 56 amino acid residues, or a polypeptide having at least 75% and more preferably at least 80% identical sequence and having GPT activity. The GS1 transgene can be encoded by polynucleotide SEQ ID NO: 3 or SEQ ID NO: 6 or a nucleotide sequence of at least 75% and more preferably at least 80% identical to SEQ ID NO: 3 or SEQ ID NO: 6 and encoding a polypeptide having GPT activity; and a nucleotide sequence encoding a polypeptide of SEQ ID NO: 4 or 7, or a polypeptide having at least 75% and more preferably at least 80% identical sequence and having GS activity.

In another specific embodiment of the present invention, the GPT transgene is a GPT polynucleotide encoding a grape derived GPT, such as a grape GPT with the sequence SEQ ID NO: 9 and SEQ ID NO: 31, and the GS transgene is a GS1 polynucleotide. The GPT transgene can be encoded by the nucleotide sequence of SEQ ID NO: 8; a nucleotide sequence of at least 75% and more preferably at least 80% identical to SEQ ID NO: 8, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding a polypeptide of SEQ ID NO: 9 or SEQ ID NO: 31, or a polypeptide having at least 75% and more preferably at least 80% identical sequence and having GPT activity.

In yet another specific embodiment of the present invention, the GPT transgene is a GPT polynucleotide encoding a Rice derived GPT, such as rice GPT with the sequences SEQ ID NO: 11 and SEQ ID NO: 32, and the GS transgene is a GS1 polynucleotide. The GPT transgene can be encoded by the nucleotide sequence of SEQ ID NO: 10; a nucleotide sequence of at least 75% and more preferably at least 80% identical to SEQ ID NO: 10, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding a polypeptide of SEQ ID NO: 11 or SEQ ID NO: 32, or a polypeptide having at least 75% and more preferably at least 80% identical sequence and having GPT activity.

In yet another specific embodiment of the present invention, the GPT transgene is a GPT polynucleotide encoding Soybean-derived GPT, such as soybean GPT with the sequences SEQ ID NO: 13, SEQ ID NO: 33 or SEQ ID NO: 33 with isoleucine at N- ends of the sequence, and the GS transgene is a GS1 polynucleotide. The GPT transgene can be encoded by the nucleotide sequence of SEQ ID NO: 12; a nucleotide sequence of at least 75% and more preferably at least 80% identical to SEQ ID NO: 12, and encoding a polypeptide having GPT activity; the nucleotide sequence encoding the polypeptide of SEQ ID NO: 13 or SEQ ID NO: 33 or SEQ ID NO: 33 with isoleucine at the N-ends of the sequence, or a polypeptide having at least 75% and more preferably at least , 80% identical sequence and having GPT activity.

In yet another specific embodiment of the present invention, the GPT transgene is a GPT polynucleotide encoding a GPT derived from barley, such as barley GPT with the sequences SEQ ID NO: 15 and SEQ ID NO: 34, and the GS transgene is a GS1 polynucleotide. The GPT transgene can be encoded by the nucleotide sequence of SEQ ID NO: 14; a nucleotide sequence of at least 75% and more preferably at least 80% identical to SEQ ID NO: 14, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding a polypeptide of SEQ ID NO: 15 or SEQ ID NO: 34, or a polypeptide having at least 75% and more preferably at least 80% identical sequence and having GPT activity.

In another specific embodiment of the present invention, the GPT transgene is a GPT polynucleotide encoding GPT derived from striped pertsina, such as striped pertsin GPT with the sequences SEQ ID NO: 17 and SEQ ID NO: 35, and the GS transgene is a GS1 polynucleotide. The GPT transgene can be encoded by the nucleotide sequence of SEQ ID NO: 16; a nucleotide sequence of at least 75% and more preferably at least 80% identical to SEQ ID NO: 16, and encoding a polypeptide having GPT activity; a nucleotide sequence encoding a polypeptide of SEQ ID NO: 17 or SEQ ID NO: 35, or a polypeptide having at least 75% and more preferably at least 80% identical sequence and having GPT activity.

In yet another specific embodiment of the present invention, the GPT transgene is a GPT polynucleotide encoding a Bamboo derived GPT, such as the GPT Bamboo SEQ ID NO: 36, and the GS transgene is a GS1 polynucleotide. The GPT transgene can be encoded by a nucleotide sequence encoding a polypeptide of SEQ ID NO: 36, or a polypeptide having at least 75% and more preferably at least 80% identical sequence and having GPT activity.

Other GPT poinucleotides suitable for use as GPT transgenes in the practice of the present invention can be obtained in various ways, as will be appreciated by those skilled in the art, tested for their ability to control the expression of GPTs with GPT activity in a recombinant expression system (i.e. E. coli (see Examples 20-23), in a temporary in planta expression system (see Example 19), or in a transgenic plant (see Examples 1-18).

Transgenic constructs / expression vectors

In order to obtain the transgenic plants of the present invention, the gene coding sequence for the desired transgene (s) must be included in the nucleic acid construct (also interchangeably referred to herein as an (transgenic) expression vector, expression cassette, expression construct or expressed genetic construct) which can control the expression of a transgenic sequence in transformed plant cells. Such nucleic acid constructs carrying the transgene (s) in question can be introduced into a plant cell or cells using a variety of methods known in the art, including, but not limited to, electroporation, DNA bombardment, or biological approaches, microinjection, or through the use of various DNA-based vectors, such as Agrobactenum tumefaciens and Agrobacterium rhizogenes vectors. Immediately after the plant is introduced into the transformed cell, the nucleic acid construct can control the expression of the transgene (s) (i.e., GPT) introduced, either temporarily or stably. Stable expression is preferred, and it is achieved through the use of transformation vectors suitable for plants that are capable of controlling the chromosome integration of the transgenic construct. Immediately after a plant cell has been successfully transformed, it can be cultured to produce a transgenic plant.

A large number of expression vectors are known for controlling the constitutive or induced expression of inserted genes in transformed plants. In addition, various temporary expression vectors and systems are known. More suitable expression vectors are selected for use in a particular gene transformation method (see below). Generally speaking, a typical plant expression vector for producing transgenic plants will contain a transgene of interest under the regulatory control of an expression promoter, a selectable marker to aid in selection of transformants, and a transcriptional terminator sequence.

More specifically, the basic elements of a nucleic acid construct for use in the preparation of transgenic plants of the present invention are: a suitable promoter capable of controlling the functional expression of transgene (s) in a transformed plant cell, transgene (s) (i.e., GPT coding sequence), operable associated with the promoter, preferably a suitable transcription termination sequence (i.e., the terminator of the nopaline synthase enzyme gene) operably linked to the transgene and sometimes other elements useful for controlling transgene expression, as well as one or more selectable marker genes suitable for selecting the desired transgenic product (i.e., antibiotic resistance genes).

Since Agrobacterium tumefaciens is the primary transformation system used to produce transgenic plants, there are many vectors designed for agrobacterial transformation. For stable transformation, agrobacterial systems use “binary” vectors that allow plasmids to be used in both E. coli and agrobacteria, and typically contain one or more selectable markers to identify transformed plants (Hellens et al., 2000 , Technical focus: A guide to Agrobacterium binary 77 vectors. Trends Plant Sci 5: 446-451). Binary vectors for use in agrobacterial transformation systems typically contain T-DNA boundaries, multiple cloning sites, replication functions for Escherichia coli and A. tumefaciens, and a selectable marker and reporter genes.

The so-called "superbinary" vectors provide higher transformation efficiency, and, as a rule, contain additional virulence genes from Ti (Komari et al., 2006, Methods Mol. Biol. 343: 15-41). Superbinary genes are typically used in plants that exhibit lower transformation efficiency, such as cereals. Such additional virulence genes include, but are not limited to, 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. Transgenic Res. 13: 593-603; Srivatanakul et al., 2000, Additional virulence genes influence transgene expression: transgene copy number, integration pattern and expression. J. Plant Physiol. 157, 685-690; Park et al., 2000, Shorter T-DNA or additional virulence genes improve Agrobacterium-mediated transformation. Theor. Appl. Genet. 101, 1015-1020; Jin et al., 1987, Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J. Bacteriol. 169: 4417-4425).

In the embodiments of the present invention provided herein (see Examples below), expression vectors are used in which the inserted transgene (s) are placed (s) under the control of the constitutive CaMV 35S promoter and RuBisCo promoter. A number of expression vectors that use the CaMV 35S and RuBsCo promoter are known and / or commercially available and / or can be obtained using specialists in this field of technology. In addition, many promoters suitable for controlling transgene expression are known and can be used in the practice of the present invention, as described below.

Plant promoters

A large number of promoters that act in plants are known in the art. When compiling GPT and GS transgenic constructs, 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 used for expression of transgenes in plants. Examples of other strong constitutive promoters include, but are not limited to, the rice actin 1 gene promoter, CaMV 19S promoter, Ti plasmid nopaline synthase gene promoter, alcohol dehydrogenase gene promoter, and sucrose synthase gene promoter.

Alternatively, in some embodiments of the present invention, it may be preferable to select a promoter based on the desired plant cells to be transformed with the transgenic construct, the desired level of transgene expression, the desired tissue and subcellular compartment for transgene expression, the target developmental stage, and the like.

For example, when expression in photosynthetic tissues and compartments is desired, a ribulose bisphosphate carboxylase (RuBisCo) gene promoter may be used. In the examples below, expressed nucleic acid constructs containing GPT and GS1 transgenes under the control of the RuBisCo tomato promoter were obtained and used in the preparation of transgenic plants or for analysis of GPT activity in planta or in E. coli.

When expression in seeds is desired, gene promoters of various proteins stored in seeds may be used. For expression in fruits, a fruit-specific promoter such as tomato 2A11 may be used. Examples of other tissue-specific promoters include lectin encoding promoters (Vodkin et al., 1983, Cell 34: 1023-31; Lindstrom et al., 1990, Developmental Genetics 11: 160-167), grain alcohol dehydrogenase 1 (Vogel et al, 1989, J. Cell. Biochem. (Suppl. 0) 13: Part D; Dennis et al., 1984, Nucl. Acids Res., 12 (9): 3983-4000), grain light-collecting complexes (Simpson, 1986, Science, 233 : 34-38; Bansal et al., 1992, Proc. Natl. Acad. Sci. USA, 89: 3654-3658), heat shock grain protein (Odell et al., 1985, Nature, 313: 810-812; Rochester et al., 1986, EMBO J., 5: 451-458), the small subunit of pea RuBP carboxylase (Poulsen et al., 1986, Mol. Gen. Genet., 205 (2): 193-200; Cashmore et al. 1983, Gen. Eng. Plants, Plenum Press, New York, pp 29-38), mannopinsynthase Ti-p lazmides and nopalin synthase of a Ti plasmid (Langridge et al., 1989, Proc. Natl. Acad. Sci. USA, 86: 3219-3223), petunia chalcon isomerase (Van Tunen et al., 1988, EMBO J. 7 (5 ): 1257-1263), bean-rich glycine protein 1 (Keller et al., 1989, EMBO J. 8 (5): 1309-1314), truncated CaMV 35s (Odell et al., 1985, supra), potato patatin ( Wenzler et al., 1989, Plant Mol. Biol. 12: 41-50), root cell (Conkling et al., 1990, Plant Physiol. 93: 1203-1211), corn zein (Reina et al., 1990, Nucl. Acids Res. 18 (21): 6426; Kriz et al., 1987, Mol. Gen. Genet. 207 (1): 90-98; Wandelt and Feix, 1989, Nuc. Acids Res. 17 (6): 2354; Langridge and Feix, 1983, Cell 34: 1015- 1022; Reina et al., 1990, Nucl. Acids Res. 18 (21): 6426), globulin-1 (Belanger and Kriz, 1991, Genetics 129: 863-872), α-tubulin (Carpenter et al., 1992 , Plant Cell 4 (5): 557-571; Uribe et al., 1998, Plant Mol. Biol. 37 (6): 1069-1078), cab (Sullivan, et al., 1989, Mol. Gen. Genet. 215 (3): 431-440), Phosphoenolpyruvate carboxylase (PEPCase) (Hudspeth and Grula, 1989, Plant Mol. Biol. 12: 579-589), R gene complex (Chandler et al., 1989, The Plant Cell 1: 1175 -1183), chalcone synthase (Franken et al., 1991, EMBO J. 10 (9): 2605-2612) and glutamine synthetase promoters (US Pat. No. 5 , 391.725; Edwards et al., 1990, Proc. Natl. Acad. Sci. USA 87: 3459-3463; Brears et al., 1991, Plant J. 1 (2): 235-244).

In addition to constitutive promoters, various inducible promoter sequences can be used in cases where it is desirable to regulate transgene expression while the transgenic plant grows, matures, blooms, etc. Examples of such inducible promoters include promoters of heat shock genes, genes responsible for protective functions (i.e., phenylalanine ammonia lyase; see, for example, Bevan et al., 1989, EMBO J. 8 (7): 899-906), genes responsible for wound healing processes (i.e., cell wall protein genes), chemically inducible genes (i.e., nitrate reductase, chitinase) and darkly-inducible genes (i.e., asparagine synthetase; see, for example, US Pat. No. 5,256,558). A number of plant nuclear genes are also 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.).

Other inducible promoters include ABA and turgor-inducible promoters, auxin-binding protein gene promoter (Schwob et al., 1993, Plant J. 4 (3); 423-432), UDP-glucose gene promoter flavonoid-glucosyl transferase (Ralston et al., 1988, Genetics 119 (1): 185-197); MPI proteinase inhibitor promoter (Cordero et al., 1994, Plant J. 6 (2): 141-150), promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (Kohler et al., 1995, Plant Mol. Biol. 29 (6): 1293-1298; Quigley et al., 1989, J. Mol. Evol. 29 (5): 412-421; Martinez et al., 1989, J. Mol. Biol. 208 (4): 551-565) and light-inducible pea glutamine synthetase plastid gene promoter (US Pat. No. 5,391,725; Edwards et al., 1990, supra).

Plant promoters used in transgenic plants are discussed in Potenza et al., 2004, In Vitro Cell. Devel Biol - Plant. 40 (1): 1-22. Synthetic plant promoters are discussed, for example, in Venter, M., 2007, Trends Plant Sci., 12 (3): 118-124.

Glutamine-phenylpyruvate-transaminase transgene (GPT)

The present invention for the first time discloses that plants contain the enzyme glutamine-phenylpyruvate transaminase (GPT), which is directly involved in the synthesis of the signal metabolite of 2-hydroxy-5-oxoproline. To date, plant-based glutamine-phenylpyruvate-transaminases with a specific function have not been described. Applicants have isolated and tested sequences encoding a GPT polynucleotide derived from certain plant and animal species, and successfully included the gene in a heterologous transgenic host plant that exhibits markedly improved growth characteristics, including faster growth, higher leaf protein content, increased glutamine synthetase activity in leaf tissues and higher rates of CO ₂ fixation.

In practicing the present invention, the GPT gene acts as one of at least two transgenes introduced into the transgenic plants of the present invention, the other being the glutamine synthetase gene (see below).

All plant species are expected to contain GPT, which is involved in the same metabolic pathway, including the biosynthesis of the signal metabolite of 2-hydroxy-5-oxoproline. Thus, in the practical implementation of the present invention, any plant gene encoding the GPT homolog or functional variants thereof can be used in the preparation of transgenic plants of the present invention. Moreover, a structural similarity was established between the different protein structures of plant GPT and the putative (and biologically active) GPT homologue from Danio rerio (striped percina) (see Example 22), therefore, other non-plant GPT homologs can be used to obtain GPT transgenes for use in obtaining transgenic plants of the present invention.

When individually compared (by BLAST alignment) with the sequence of the mature Arabidopsis protein, SEQ ID NO: 30, the following sequence homology and identity (BLAST "positivity", including similar amino acids) were observed for the following sequences of the mature GPT protein:

[SEQ ID] Origin % Identity % positive [31] Grape 84 93 [32] Rice 83 91 [33] Soybean 83 93 [34] Barley 82 91 [35] Striped pepper 83 92 [36] Bamboo 81 90 Corn 79 90 Castor oil 84 93 Poplar 85 93

Underscoring the conservative nature of the structure of the GPT protein for most plant species, the conservative structure observed for the above plant species has been maintained for the non-human putative GPTs from striped pepper and Chlamydomonas. In the case of striped pepper, the degree of identity is very high (83% identity of the amino acid sequence of mature Arabidopsis GPT with the sequence of SEQ ID NO: 30, and 92% homology, taking into account similar amino acid residues). Mature GPT of striped pepper was confirmed by expression in E. coli, and biological activity (synthesis of 2-oxoglutaramate) was demonstrated. In order to determine whether putative GPT homologs are suitable for producing transgenic plants of the present invention with enhanced growth, it is first necessary to express their coding sequence in E. coli or another suitable host, and to determine whether the signal metabolite 2-oxoglutaramate is synthesized at elevated levels ( see Examples 19-23). If such an increase is observed, the coding sequence can then be introduced into both the homologous host plant and the heterologous host plant, and growth characteristics are evaluated. For this purpose, any analysis can be used during which it is possible to specifically determine 2-oxoglutaramate, including, but not limited to, NMR and HPLC analyzes described in Example 2 below. In addition, assays can be used that directly measure GPT activity, such as the GPT activity analysis described in Example 7.

Any plant GPT with 2-oxoglutaramate synthesis activity can be used to transform plant cells in order to obtain the transgenic plants of the present invention. It turns out that there is a high level of structural homology among plant species, which, as it turns out, covers dicotyledonous plants, which is evident due to the close homology between different plant GPT proteins and the supposed GPT homolog from striped percina. Therefore, various plant GPT genes can be used to produce transgenic plants with enhanced growth for a variety of heterologous plant species. In addition, GPT transgenes expressed in a homologous plant are also expected to lead to the desired characteristics of enhanced growth (i.e., rice glutamine transaminase is overexpressed in transgenic rice plants), although it is possible that regulation within the homologous cell may weaken the transgene expression in some way, what might not happen in a heterologous cell.

Glutamine synthetase transgene (GS):

In practicing the invention, the glutamine synthetase (GS) gene functions as one of the at least two transgenes included in the transgenic plants of the present invention (the GPT being the second of two transgenes).

Glutamine synthetase plays a key role in the metabolism of nitrogen in plants, as well as animals and bacteria. The GS enzyme catalyzes the addition of ammonia to glutamate with the synthesis of glutamine in an ATP-dependent reaction. GS enzymes from different species show highly conserved amino acid residues, which are considered important for the function of the active site, which indicates that GS enzymes act in a similar way (see Eisenberg et al., Biochimica et Biophysica Acta, 1477: 122 145 , 2000).

GS is distributed in various subcellular positions (chloroplast and cytoplasm) and is found in various plant tissues, including leaf, root, shoot, seeds and fruits. There are two main isoforms of plant GS: the cytoplasmic isoform (GS1) and the plastid (chloroplast) isoform (GS2). GS2 is mainly found in leaf tissue and is involved in the assimilation of ammonia produced by photorespiration or by reduction of nitrate. GS1 is mainly found in leaf and root tissues, usually exists in a number of different isoforms in higher plants, and is involved in the assimilation of ammonia obtained in 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 Mot. 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). Many GS genes are associated with a complex set of promoters that are able to express GS in an organ and tissue in a specific way, as well as in a manner dependent on environmental factors.

Plant glutamine synthetase consists of eight subunits, and the native enzyme in plants has a molecular weight in the range of 320 to 380 kDa, with each subunit having a molecular weight in the range of 38 to 45 kDa. The GS1 genes of some plants, especially leguminous plants, were cloned and ordered (Tischer et al., 1986, Mol Gen Genet. 203: 221-229; Gebhardt et al., 1986, EMBO J. 5: 1429-1435; Tingey et al., 1987, EMBO J. 6: 1-9; Tingey et al., 1988, J Biol Chem. 263: 9651-9657; Bennett et al., 1989, Plant Mol Biol. 12: 553-565; Boron and Legocki, 1993, Gene 136: 95-102; Roche et al., 1993, Plant Mol Biol. 22: 971-983; Marsolier et al., 1995, Plant Mol Biol. 27: 1-15; Temple et al., 1995, Mol Plant-Microbe Interact. 8: 218-227). All have been found to be encoded by nuclear genes (see, Morey et al., 2002, Plant Physiol. 128 (1): 182-193).

Chloroplastic GS2 appears to be encoded by a single gene, while the various cytoplasmic isoforms of GS1 are encoded by multi-gene families (Tingey et al., 1987, supra; Sakamoto et al., 1989, Plant Mol. Biol. 13: 611-614; 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, above). GS1 multigene families, as it turns out, encode various subunits that can combine to form homo or heteroctamers, and different members exhibit a unique expression model, suggesting that these members, which are genes, are regulated in different ways, which may be associated with different the functional roles that glutamine synthetase plays in the overall metabolism of nitrogen (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., 19 95, supra; Temple et al., 1995, supra; Dubois et al., 1996, supra).

In one embodiment of the present invention, the sequence encoding the GS1 gene is used to obtain GS transgenic constructs. In specific embodiments of the present invention, further described in the examples below, the sequence encoding the Alfalfa or Arabidopsis GS1 gene is used to obtain a transgenic construct that can be used to obtain a transgenic plant expressing a GS1 transgene. As an example, such a design can be used to transform agrobacteria. Transformed agrobacteria are then used to obtain T ₀ transgenic plants. Example 5 demonstrates the preparation of T ₀ GS1 transgenic tobacco plants using this approach. Similarly, Examples 6 and 17 demonstrate the production of T ₀ GS1 transgenic tomato plants, Example 8 demonstrate the production of T ₀ GS1 transgenic pepper plants, Examples 9 and 10 demonstrate the production of T ₀ GS1 transgenic bean plants, Examples 11 and 12 demonstrate the production of T ₀ GS1 transgenic cowpea plants, Example 13 demonstrates the preparation of T ₀ GS1 transgenic alfalfa, Example 14 demonstrates the preparation of T ₀ transgenic plants GS1 cantaloupe, Example 15 demonstrates the preparation of T ₀ GS1 transgenic plants of pumpkin, Example 16 demonstrates the preparation of T ₀ GS1 tr nsgennyh plant Arabidopsis, and Example 18 demonstrates the preparation of T ₀ transgenic plants GS1 cantaloupe.

Transcription Terminators:

In preferred embodiments of the present invention, a 3 'transcription termination sequence is inserted downstream of the transgene in order to control transcription termination and allow the polyadenylation of the transcript mRNA to be corrected. Suitable transcription terminators are those that are known to function in plants, including, but not limited to, the Nopalin synthase (NOS) and octopin synthase (OCS) genes of Agrobacterium tumefaciens, the T7 transcript from the octopin synthase gene, the 3 'end of the inhibitor I gene II from potato or tomato, CaMV 35S terminator, tml terminator and rbcS E9 terminator from peas. In addition, a natural gene transcription terminator can be used. In specific embodiments of the present invention described using the examples below, a nopaline synthase transcription terminator is used.

Selection Markers:

Selection markers are typically introduced into transgenic expression vectors to provide means for selecting transformants. While different types of markers are available, various negative selection markers are generally used, including those that confirm resistance to a selection agent that inhibits or kills untransformed cells, as important genes are resistant to antibiotics (such as kanamycin, gentamicin, anamycin , hygromycin and hygromycin B) or are resistant to herbicides (such as sulfonylurea, glufosinate, phosphinotricin and glyphosate). Screenable markers include, for example, genes encoding β-glucuronidase (Jefferson, 1987, Plant Mol. Biol. Rep 5: 387-405), genes encoding luciferase (Ow et al., 1986, Science 234: 856-859) and various genes encoding proteins involved in the production or regulation of anthocyanin pigments (See, for example, US patent 6,573,432). E. coli glucuronidase gene (gus, gusA or uidA) has become a widely used selection marker in plant transgenics, mainly due to the stability of the glucuronidase enzyme, high sensitivity and easy detection (e.g. fluorometric method, spectrophotometric method and various histochemical methods). Moreover, there is essentially no detectable glucuronidase in most species of higher plants.

Transformational methods and systems:

Various methods for introducing transgenic expression vector constructs of the present invention into plants and plant cells are well known to those skilled in the art, and any methods suitable for transforming target plants and plant cells can be used.

Agrobacterium-mediated transformation is arguably the most common method used in plant transgenics, and the protocols of Agrobacterium-mediated transformation of a large number of plants are widely described in the literature (see, for example, Agrobacterium Protocols, Wan, ed., Humana Press, 2 ^nd edition, 2006). Agrobacterium tumefaciens is a gram-negative soil bacterium that leads to the formation of tumors (root cancer disease) in a large number of dicotyledonous species, through the insertion of a small segment of tumor-inducing DNA (T-DNA ", 'transferred DNA') into the plant cell, which randomly integrates into the plant’s genome, and which ultimately can become stably integrated into the plant’s genome. Directly repeating DNA sequences, called T-DNA boundaries, define the left and right ends of T-DNA. T-DNA can be physically separated from the rest of the Ti plasmid to give a 'binary vector' system.

Agrobacterial transformation can be used for stably transforming dicotyledonous plants, monocotyledonous plants and their cells (Rogers et al., 1986, Methods Enzymol., 118: 627-641; Hernalsteen et al., 1984, EMBO J., 3: 3039-3041; Hoykass-Van Slogteren et al., 1984, Nature, 311: 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). Various methods for introducing DNA into Agrobacteria are known, including electroporation, freeze / thaw methods, and tripartite crosses. The most efficient way to place foreign DNA in Agrobacterium is through electroporation (Wise et al., 2006, Three Methods for the Introduction of Foreign DNA into Agrobacterium, Methods in Molecular Biology, vol. 343: Agrobacterium Protocols, 2 / e, volume 1; Ed ., Wang, Humana Press Inc., Totowa, NJ, pp. 43-53). In addition, given that a large percentage of T-DNA does not integrate, Agrobacterium-mediated transformation can be used to obtain transient transgene expression through the transcriptional ability of unincorporated transgenic construct molecules (Helens et al., 2005, Plant Methods 1:13).

A large number of vectors and methods for agrobacterial transformation have already been described (Karimi et al., 2002, Trends Plant Sci. 7 (5): 193-5), and many such vectors can be obtained commercially (for example, Invitrogen, Carlsbad, CA). In addition, the number of available “open sources” of agrobacterial transformation vectors is constantly increasing (for example, pCambia vectors; Gambia, Canberra, Australia). See also the sub-chapter “Transgenic constructs” also cited in this document. In a specific embodiment of the present invention, described further in the examples, the pMON316-based vector was used in sheet metal transformation systems according to Horsch et. al. (Horsch et al., 1995, Science 227: 1229-1231) to produce transgenic tobacco and tomato plants with enhanced growth.

Other well-known transformation methods that can be used to obtain the transgenic plants of the present invention include, but are not limited to, microprojection bombardment or biolistic transformation methods, protoplast transformation of bare DNA with calcium, polyethylene glycol (PEG) or electroporation (Paszkowski et al., 1984 , EMBO J. 3: 2727-2722; Potrykus et al., 1985, Mol. Gen. Genet. 199: 169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82: 5824-5828 ; Shimamoto et al., 1989, Nature, 338: 274-276.

Biolistic transformation involves the injection of millions of metal particles coated with DNA into target cells or tissues using a biolistic device (or “gene gun”), some of which are commercially available. Inside the cell, DNA elutes from the particles, and part can be stably incorporated into one or more cell chromosomes (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. Pena, Humana Press Inc., Totowa, NJ).

Electroporation is a technique that uses short high-intensity electric fields to reversibly permeabilize 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. Pena, 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. 351-366; Joersbo and Brunstedt, 1991, Electroporation: mechanism and transient expression, sTa6nuu, a transformation and biological effects in plant protoplasts. Physiol. Plant. 81, 256-264; Bates, 1994, Genetic transformation of plants by protoplast ele ctroporation. Mol. Biotech. 2: 135-145; Dillen et al., 1998, Electroporation-mediated DNA transfer to plant protoplasts and intact plant tissues for transient gene expression assays, in Cell Biology, Vol. 4, ed., Cells, Academic Press, London, UK, pp. 92-99). The essence of the technique is to create water pores in the cell membrane that are large enough to allow DNA molecules (and other macromolecules) to enter the cell, where a transgenic expression construct (like T-DNA) can be stably incorporated into plant genomic DNA , resulting in transformed cells from which transgenic plants can be obtained.

New transformation methods include the so-called "floral dip" methods, which provide ease of use without requiring plant tissue culture requirements, as is the case with other commonly used transformation techniques (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 are not widely used for a wide range of different plant species. Briefly, the transformation of "floral dip" is carried out by dipping flowering plants into the corresponding strain of Agrobacterium tumefaciens or spraying this strain on flowering plants. Seeds collected from these T ₀ plants are then grown with selection to identify transgenic T ₁ plants. Example 16 shows a floral dip inoculation of Arabidopsis to produce transgenic Arabidopsis plants.

Other transformation methods include those in which developing seeds or seedlings of plants are transformed using vectors such as agrobacterial vectors. For example, as shown in Example 8, such vectors can be used to transform developing seeds by injecting a suspension or mixture of a vector (i.e., Agrobacteria) directly into the seed chamber of developing pods (i.e., pepper pods, bean pods, pea pods and the like) . Seedlings can be transformed as described for Alfalfa in Example 13. Germinating seeds can be transformed as described for Camelina in Example 18. Intrauterine methods in which the vector is introduced by injection into the fetus or developing fetus can be applied as described for the Cantaloupe melon in Example 14 and for the pumpkin ordinary in Example 15.

Still other transformation methods include those in which the flower structure is a target for inoculating the vector, such as the flower inoculation methods described for beans in Examples 9 and 10, peas in Examples 11 and 12, and tomato in Example 17.

The above plant transformation techniques can be used to introduce transgenes into a number of different plant cells and tissues, including, but not limited to, whole plants, explants (organs and tissues), including chloroplasts, flowering tissues and cells, protoplasts, meristem cells, callus, immature embryos and gametes, such as microspores, pollen, sperm and eggs, tissue cultured cells of any of the foregoing, any other cells from which a fertile regenerated transgenic plant can be obtained. Kalus originates from tissue sources, including, but not limited to, immature embryos, apical seedling meristems, microspores, and the like. Cells capable of proliferation like callus are also recipient cells for genetic transformation.

Methods for regenerating plants from transformed plant cells, tissues or organs are well known and described for many different types of plants.

As illustrated, transformed germs (obtained from transformed cells or tissues) are cultured in a nutrient medium suitable for the development of the root system, supplemented with a selective agent used in the transformation (that is, an antibiotic such as kanamycin. Immediately after the development of the root system, the transformed plants transfer into the soil and give them the opportunity to grow to the state of a mature plant.When flowering, mature plants are preferably self-pollinated (self-fertilized), and I collect the seeds t and are used to produce subsequent generations Examples 3-6 show regeneration of transgenic tobacco and tomato plants.

T ₀ transgenic plants can be used to obtain subsequent generations (for example, T ₁ , T ₂ , etc.) by self-pollination of primary or secondary transformants, or by sexually mating primary or secondary transformants with other plants (transformed or non-transformed). For example, as described in Example 7 below, individual plants overexpressing the Alfalfa GS1 gene and having improved characteristics compared to wild-type plants were crossed with individual plants overexpressing the Arabidopsis GPT gene and having improved characteristics compared to wild-type plants, by simple sexual intercourse, using manual pollen transfer. Reciprocal crosses were made in such a way that each plant served as a male in one row of crosses, and each plant served as a female in the second row of crosses. During the stage of plant growth to a mature state, plants are usually examined for a growth phenotype, CO ₂ fixation rate, etc. (see next section).

Selection of transgenic plants with enhanced growth:

Transgenic plants can be selected, screened, and can be characterized using standard techniques. Preferred transgenic plants of the present invention exhibit one or more phenotypic characteristics indicating enhanced growth and / or other desirable agronomic characteristics. Transgenic plants are typically regenerated at selective pressure in order to select transformants before creating the next generation of transgenic plants. In addition, the selective pressure used can be applied to generations following the T ₀ generation to ensure the presence of the desired transgenic expression construct or cassette.

T ₀ transformed plant cells, calluses, tissues or plants can be identified and isolated by selection and screening for a set of genes and / or phenotypic characteristics encoded by marker genes contained in the transgenic expression construct used for transformation. For example, selection can be carried out by growing potentially transformed plants, tissues or cells in a nutrient medium containing an overwhelming amount of an antibiotic or herbicide to which the transforming genetic construct can provide resistance. In addition, transformed plant cells, tissues, and plants can be identified by screening for activity of marker genes (i.e., β-glucuronidase) that may be present in the transgenic expression construct.

Various physical and biochemical methods that can be used to identify plants containing the desired transgenic expression construct are also known. Examples of such methods include Southern blot analysis or various methods for amplifying nucleic acids (i.e., PCR) to identify a transgene, transgenic expression construct or its elements, Northern blotting, S1 ribonuclease analysis, PCR amplification with reverse transcriptase (RT-PCR) for for detecting and determining RNA of transcription products and gel electrophoresis of proteins, Western blotting, immunoprecipitation, enzyme immunological analysis and similar methods can be used to identify encoded proteins and expressed by a transgene.

In another approach, expression levels of genes, proteins and / or metabolic compounds that are known to be modulated by transgene expression in a target plant can be used to identify transformants. In one embodiment of the present invention, elevated levels of the signal metabolite of 2-oxoglutaramate can be used for screening for the desired transformants, as shown in the examples. Similarly, elevated levels of GPT and / or GS activity can be evaluated, as shown in the Examples.

Finally, the transformed plants of the present invention can be screened for enhanced growth and / or other desirable agronomic characteristics. In fact, some degree of phenotypic screening is generally desirable for identifying transformed lines with higher growth rates, higher seed yields, etc., especially when plants are selected for subsequent selfing, crossbreeding and backcrossing. For this purpose, various parameters can be applied, including, but not limited to, growth rates, total wet tissue mass, dry weight, seed and fruit yield (quantity, weight), seed and / or seed box sizes, seed box yield (e.g. , quantity, mass), leaf sizes, plant sizes, increased flowering, time period before flowering, total protein content (in seeds, fruits, plant tissues), specific protein content (i.e., GS), nitrogen content, free amino acid and specific metabolic levels Cesky compounds (ie, 2-oxoglutaramate). In general, these phenotypic measurements are compared to those obtained for a parental identical or similar plant line, an untransformed identical or similar plant, or an identical or similar wild-type plant (i.e., normal or parent compound). Preferably, at least initially, measurements of the selected phenotypic characteristic (s) in the target transgenic plant are carried out in parallel with the measurement of the same characteristic (s) in the normal or parent compound. Typically, many plants are used to establish the phenotypic attractiveness and / or phenotypic superiority of a transgenic plant with respect to any particular phenotypic characteristic.

Preferably, primary transformants are selected, and then they are used to obtain T ₁ and subsequent generations by self-pollination (self-fertilization), until the transgenic genotype retains the ability to transmit the trait to offspring (that is, the plant is homozygous for the transgene). In fact, this is done by screening each generation for the desired traits and self-pollination of plants in which the desired traits were detected, often repeatedly (i.e. 3 or 4 generations). As shown herein, transgenic plant lines obtained by propagation by at least one sexual cross (Tobacco, Arabidopsis, Tomato) exhibit higher transgenic product activity compared to lines which were not obtained by sexual reproduction and the concomitant increase the number of copies of the transgene.

Stable transgenic lines can be crossed and backcrossed to produce varieties with any number of desirable traits, including traits laid by transgenes, multiple copies of the transgene, etc. In addition, stable transgenic plants can then be genetically modified by transforming such plants with the following transgenes or additional copies of the original transgene. Also considered are transgenic plants obtained through a single transformation event during which multiple copies of a given transgene or multiple transgenes are introduced. Various conventional propagation methods are well known to those skilled in the art (see, for example, Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987)).

As another object, the present invention discloses transgenic plants characterized by increased nitrogen utilization efficiency. The nitrogen utilization efficiency can be expressed as the yield of a plant for a given amount of nitrogen. In the examples provided herein, all transgenic and control plants received the same nitrogen solutions in the same amounts. All transgenic plants had a higher yield, and thus had a higher nitrogen utilization efficiency.

As another object of the present invention discloses transgenic plants and their seeds with increased tolerance to high salt growth conditions. This object of the present invention is shown in Example 24, which describes the germination of seeds of a transgenic tobacco plant under conditions with a very high salt content (200 mm NaCl). While the seeds of a similar wild-type tobacco plant sprouted at an average rate of only about 10%, the seeds of a transgenic tobacco plant achieved almost the same germination rate that is achieved under salt-free conditions by both the seeds of transgenic plants and the seeds of wild-type plants. or about 92%.

EXAMPLES

Various objects of the present invention are further described and illustrated by way of examples, which, however, are not intended to limit the scope of the present invention.

EXAMPLE 1: Isolation of the gene for glutamine-phenylpyruvate transaminase (GPT) from ARABIDOPSIS:

In an attempt to locate a plant enzyme that is directly involved in the synthesis of the 2-oxoglutaramate signal metabolite, the applicants suggested that the putative plant enzyme may have some degree of structural affinity with the human protein, which was characterized as participating in the synthesis of 2-oxoglutaramate. The human protein, glutamine transaminase K (E.C. 2.6.1.64) (also referred to in the literature as β-lyase cysteine conjugate, kinurenine aminotransferase, glutamine-phenylpyruvate transaminase and under other names) has been shown to be involved in the processing of halogenated cysteine conjugates xenobiotics (Perry et al., 1995, FEBS Letters 360: 277-280). However, human β-lyase cysteine conjugate is more likely to exhibit detoxifying activity in humans and animals than it has activity associated with participation in nitrogen assimilation (Cooper and Meister, 1977, supra). Nevertheless, the possible participation of this protein in the synthesis of 2-oxoglutaramate is of great interest.

Using the protein sequence of the human β-lyase cysteine conjugate, a search in the Arabidopsis TIGR database of protein sequences revealed one potentially related sequence, a polypeptide encoded by a partial sequence of the Arabidopsis locus at At1q77670, with about 36% of the sequence having homology / identity for the aligned regions.

The complete coding region of the gene was then amplified from the Arabidopsis (Stratagen) cDNA library using the following pair of primers:

5'-CCC ATCGAT GTACCTGGACATAAATGGTGTGATG-3 '[SEQ ID NO: 37]

5'-GAT GGTACC TCAGACTTTTCTCTTAAGCTTCTGCTTC-3 '[SEQ ID NO: 38]

These primers are designed to incorporate Cla I ( ATCGAT ) and Kpn I ( GGTACC ) restriction sites to facilitate subcloning of the sequence into expression vectors to generate transgenic plants. The Takara ExTaq DNA polymerase enzyme was used for high-precision PCR under the following conditions: initial denaturing at 94 ° C for 4 minutes, 30 cycles at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, elongation at 72 ° C for 90 seconds, final elongation at 72 ° C for 7 minutes. The amplification product was subjected to the action of Cla I and Kpn I restriction enzymes, isolated by agarose gel electrophoresis, and ligated into the pMon316 vector (Rogers, et. Al. 1987 Methods in Enzymology 153: 253-277), which contains the 35S constitutive promoter of the mosaic virus cauliflower (CaMV, also CMV) and 3 'nopalin synthase terminator (NOS). The ligation product was transformed into DH5α cells, and transformant sequences were established to verify insertion.

cDNA size of 1.3 thousand pairs of nucleotides (kb) was isolated, and its sequence was determined, and found that it encodes a full-length protein of 440 amino acids in length, including the putative chloroplast signal sequence.

EXAMPLE 2: Obtaining biologically active recombinant ARABIDOPSIS glutamine-phenylpyruvate transaminase (GPT):

In order to verify whether the protein encoded by the cDNA isolated as described in Example 1 above is capable of catalyzing the synthesis of 2-oxoglutaramate, cDNA was expressed in E. coli, purified and its ability to synthesize 2-oxoglutaramate was studied using the standard method.

NMR analysis of 2-oxoglutaramate:

Briefly, the resulting purified protein was added to a reaction mixture containing 150 mM Tris-HCl, pH 8.5, 1 mM beta-mercaptoethanol, 200 mM glutamine, 100 mM glyoxylate and 200 μM pyridoxal-5'-phosphate. The reaction mixture, without the addition of a test protein, was used as a control. The test and control reaction mixtures were incubated at 37 ° C for 20 hours, and then clarified by centrifugation to remove precipitated material. Supernatants were analyzed for the presence and amount of 2-oxoglutaramate using ¹³ C NMR with an authentic chemically synthesized 2-oxoglutaramate as a control. The reaction products are 2-oxoglutaramate and glycine, while the amount of substrates (glutamine and glyoxylate) is greatly reduced. Cyclic 2-oxoglutaramate gives a separate signal that can be easily distinguished from an open-chain glutamine precursor.

HPLC analysis of 2-oxoglutaramate:

In an alternative analysis of GPT activity, HPLC is used to determine the presence of 2-oxoglutaramate, following a modification of Calderon et al., 1985, J Bacteriol 161 (2): 807-809. Briefly, the modified extraction buffer contains 25 mM Tris-HCl pH 8.5, 1 mM EDTA, 20 μM FAD, 10 mM Cysteine and about 1.5% (v / v) mercaptoethanol. Tissue samples taken from the test material (i.e., plant tissue) were added to the extraction buffer at a ratio of about 1/3 (w / v), incubated for 30 minutes at 37 ° C, and the reaction was stopped with 200 μl of 20% TCA After about 5 minutes, the analyzed mixture was centrifuged, and the supernatant was used to determine the amount of 2-oxoglutaramate by HPLC using an ION-300 7.8 mm ID X 30 cm L column, with a mobile phase of 0.01 N H ₂ SO ₄ , flow rate of about 0.2 ml / min, at 40 ° C. The injection volume was about 20 μl, and the retention time was 38 to 39 minutes. Detection was carried out using UV radiation with a wavelength of 210 nm.

Results of NMR analysis:

This experiment allowed us to determine that the test protein is capable of catalyzing the synthesis of 2-oxoglutaramate. Therefore, these data indicate that the isolated cDNA encodes glutamine-phenylpyruvate transaminase, which is directly involved in the synthesis of 2-oxoglutaramate in plants. Accordingly, the test protein can be designated as Arabidopsis glutamine-phenylpyruvate-transaminase or "GPT".

The nucleotide sequence of the sequence encoding Arabidopsis GPT. shown in the Table of sequences, SEQ ID NO. 1. The translated amino acid sequence of the GPT protein is shown as SEQ ID NO. 2.

EXAMPLE 3: Creation of transgenic tobacco plants overexpressing ARABIDOPSIS GPT:

The creation of the expression plant vector pMON-PJU:

Briefly, 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 pMON316 vector, which contains the GPT gene under the control of the constitutive 35S cauliflower mosaic virus (CaMV) promoter and the nopalin synthase transcription terminator. Kanamycin resistance gene is included to provide a selectable marker

Agrobacterium-mediated plant transformations:

pMON-PJU and the control vector pMon316 (without DNA insertion) were transferred to the pTiTT37ASE Agrobacterium tumefaciens strain using the standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9: 155-159), followed by application of spectinomycin antibiotics onto LB plates (100 μg / ml) and kanamycin (50 μg / ml). Antibiotic-resistant colonies of agrobacteria were examined by PCR for plasmid content.

Tobacco plants (Nicotiana tabacum cv. Xanthi) were transformed using pMON-PJU transformed agrobacteria using a leaf disc transformation system according to Horsch et. al. (Horsch et al., 1995, Science 227: 1229-1231). Briefly, sterile leaf discs were inoculated and cultured for two days, then transferred to selective MS medium containing 100 mg / ml kanamycin and 500 mg / ml clafaran. Transformants were confirmed by their ability to form roots in a selective medium.

Obtaining GPT of transgenic tobacco plants:

Callus was obtained from sterile leaf segments on a Murashige & Skoog (M&S) medium from which transformant sprouts emerged. These sprouts were then transferred to a selection medium that allows the formation of the root system (M&S medium with kanamycin as a selection tool). Healthy transformed tobacco plants with a developed root system were then transferred to the soil and given them the opportunity to develop to a mature state, when flowering plants were self-pollinated, and the seeds were collected. In the process of the growth stage, plants were examined for growth phenotype and CO ₂ fixation rate for many young transgenic plants.

Obtaining T1 and T2 generation of GPT transgenic plants:

Seeds collected from the T ₀ generation of transgenic tobacco plants germinated on M&S medium containing kanamycin (100 mg / L) to determine the transgene. At least every fourth seed did not germinate on this medium (kanamycin is expected to inhibit the germination of seeds without resistance that could be obtained by normal genetic segregation of the gene), and more than half of the germinating seeds were excluded, since they showed sensitivity (even moderate) to kanamycin.

Surviving plants (T ₁ generation) grew rapidly, and these plants self-pollinated to produce seeds for T ₂ generation. Seeds from T ₁ generation sprouted on MS medium supplemented with kanamycin (10 mg / L) to determine the lines of transformants. After 14 days, they were transferred to sand with the addition of a quarter-concentration Hoagland nutrient solution with 25 mM potassium nitrate. The seeds were allowed to grow at 24 ° C with a photoperiod of 16 hours of light and 8 hours of darkness, with a light intensity of 900 micromoles per square meter per second. Harvested 14 days after transfer to the sand.

Characteristic GPT of transgenic plants:

Collected transgenic plants (both GPT transgenes and vector control transgenes) were analyzed for glutamine synthetase activity in the root and leaf, the raw tissue mass of the whole plant, the total protein in the root and leaf, and the CO ₂ fixation rate (Knight et al., 1988, Plant Physiol. 88: 333). Non-transformed wild-type A. tumefaciens plants were analyzed by the same parameters in order to establish a baseline control.

The growth characterization results are shown in Table I below. Additionally, a GPT photograph of a transgenic plant compared to a wild-type control plant is shown in FIG. 2 (for a GS1 transgenic tobacco plant, see Example 5). For all parameters analyzed, GPT transgenic tobacco plants showed improved growth characteristics. In particular, GPT transgenic plants showed an increase in the rate of CO ₂ fixation _of more than 50%, and an increase in glutamine synthetase activity in leaf tissue by more than a factor of two compared to control plants of the wild type. In addition, the GS: leaf: root ratio increased almost three-fold in transaminase transgenic plants compared to the wild-type control. Raw tissue mass and total protein increased in transgenic plants by about 50% and 80% (leaf), respectively, relative to the wild-type control. These data demonstrate that tobacco plants overexpressing the Arabidopsis GPT transgene achieve significantly enhanced growth and an increased rate of CO ₂ fixation.

Table I Protein mg / g wet tissue mass Sheet Root Wild type control 8.3 2.3 Line PN1-8 second control 8.9 2.98 PN9-9 line 13.7 3.2

Glutamine synthetase activity, micromol / min / mg protein Wild type (Leaf: root ratio = 4.1: 1) 4.3 1.1 PN1-8 (Leaf: root ratio = 4.2: 1) 5.2 1.3 PN9-9 (Leaf: root ratio = 10.9: 1) 10.5 0.97 The total mass of raw plant tissue, g Wild type 21.7 PN1-8 26.1 PN9-9 33.1 The fixation rate of CO ₂ , µmol / m ² / s Wild type 8.4 PN1-8 8.9 PN9-9 12.9 Data are shown as the average of three plants. Wild type (WT) - control plants; unregenerated or non-transformed. PN1 lines were obtained by regeneration after transformation, using the design without insertion of the gene. Control in relation to the processes of regeneration and transformation. PN 9 lines were obtained by regeneration after transformation using the construct with the Arabidopsis GPT gene.

EXAMPLE 4: Obtaining transgenic tomato plants bearing the ARABIDOPSIS GPT transgene:

Transgenic tomato plants of Lycopersicon esculentum (Micro-Tom Tomato) bearing the Arabidopsis GPT transgene were obtained using the vectors and methods described in Example 3. T ₀ transgenic tomato plants were generated and grown to maturity. Data on the characteristics of the initial GPT growth of transgenic tomato plants are shown in Table II. Transgenic plants showed a significant increase in growth rate, increased flowering, and an increase in seed yield compared to control plants of the wild type. In addition, transgenic plants developed many major stems, while wild-type plants developed only one main stalk. A GPT photograph of a transgenic tomato plant compared to a wild-type plant is shown in FIG. 3 (for GS1 transgenic tomato plants, see Example 6).

TABLE II Growth characteristics Wild type tomato plant GPT Transgenic Tomato Plant Stem height, cm 6.5 18, 12, 11 main stems The stalks one 3 main, 0 others Kidney 2 16 Flowers 8 12 Fruit 0 3

EXAMPLE 5: Obtaining transgenic tobacco plants overexpressing ALFALFA GS1:

Obtaining a plant expression vector pGS111:

Transgenic tobacco plants overexpressing the Alfalfa GS1 gene were obtained as previously described (Temple et al., 1993, Mol. Gen. Genetics 236: 315-325). Briefly, the plant expression vector pGS111 was composed by inserting a full-length coding sequence, along with extended 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) in pMON316 (Rogers et al., 1987, supra), placing the transgene under the control of the constitutive 35S promoter of cauliflower mosaic virus (CaMV) and nopalin synthase (NOS) transcription terminator. The kanamycin resistance gene was included to provide a selectable marker.

Getting GS1 transformants:

pGS111 was transferred to the Agrobacterium tumefaciens pTiTT37ASE strain using triple crosses as described (Rogers et al., 1987, supra; Unkefer et al., US Patent No. 6,555,500). Nicotiana tabacum cv. Xanthi was transformed with pGS111 by transformed agrobacteria using a leaf disc transformation system according to Horsch et. al. (Horsch et al., 1995, Science 227: 1229-1231). Transformants were subjected to selection and regeneration on MS medium containing 100 μg / ml kanamycin. The root system of the germs developed in the same medium (with kanamycin, without hormones), and then the germs were transferred to the soil medium: perlite: vermiculite (3: 1: 1), grown to maturity and provided the possibility of self-pollination. The seeds of this T ₀ generation were harvested, and subsequent generations were obtained by self-pollination and subsequent selection using kanamycin. The best growth plants were used to produce T3 offspring for crosses with the best GPT overexpressing lines determined as shown in Example 3. A GS1 photograph of the transgenic plant and its comparison with the wild-type control plant are shown in FIG. 2 (together with the GPT transgenic tobacco plant, see Example 3)

EXAMPLE 6: Obtaining transgenic tomato plants bearing ALFALFA GS1 transgene:

Transgenic tomato plants of Lycopersicon esculentum (Micro-Tom Tomato) carrying the Alfalfa GS1 transgene were obtained using the vector described in Example 5, and the transformation protocol is essentially disclosed in (Sun et al., 2006. Plant Cell Physiol. 46 (3) 426-31). T ₀ transgenic tomato plants were generated and grown to maturity. Data on the characteristics of the initial GPT growth of transgenic tomato plants are presented in Table III. Transgenic plants showed a significant increase in growth rate, accelerated flowering, and increased yield compared to control wild-type plants. In addition, transgenic plants developed many major stems, while wild-type plants developed only one main stalk. A GS1 photograph of a transgenic tomato plant, compared to a wild-type plant, is shown in FIG. 3 (for GPT transgenic tomato plants, see Example 4).

TABLE III Growth characteristics Wild type tomato plant GS1 Tomato Transgenic Plant Stem height, cm 6.5 16, 7, 5 main stems The stalks one 3 major, 3 medium, 1 small Kidney 2 2 Flowers 8 13 Fruit 0 four

EXAMPLE 7: Obtaining transgenic tobacco plants bearing GS1 and GPT transgenes:

In an attempt to determine whether a combination of GS1 and GPT transgenes in one transgenic plant can lead to such improvements in which growth and other agronomic characteristics can be enhanced, a series of sex crossings were carried out between highly productive lines of transgenic plants with one transgene (GS1 or GPT). The results obtained are simply amazing, since these crosses regenerated offspring plants that have amazing and previously not observed increases in growth rates, biomass yield and seed yield.

Materials and methods:

Transgenic tobacco plants with a single transgene, overexpressing GPT or GS, 1 received, as described in Examples 3 and 4, respectively. Several of the fastest growing GPT lines of transgenic T2 generation plants were crossed with the fastest growing GS1 lines of transgenic T3 generation plants using reciprocal crosses. The offspring were then selected on kanamycin-containing M&S medium as described in Example 3, and plant growth, flowering, and seed yield were examined.

Tissue extracts for determining GPT and GS activities: tissue extract for determining GPT activity was extracted from crude plant tissue after grinding in 100 mM cold Tris-HCl, pH 7.6, containing 1 mM ethylenediaminetetraacetic acid, 200 mM pyridoxalphosphate and 6 mM mercaptoethanol in a ratio of: 3 ml per gram of tissue. The extract was clarified by centrifugation and used in the analysis. Tissue extract for determining GS activity was extracted from crude plant tissue after grinding in 50 mM cold imidazole, pH 7.5, containing 10 mM MgCl2 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 analysis. GPT activity was analyzed as described in Calderon and Mora, 1985, Journal of Bacteriology 161: 807-809. GS activity was measured as described in Shapiro and Stadtmann, 1970, Methods in Enzymology 17A: 910-922. Both assays included incubation with substrates and cofactor at the appropriate pH. Detection was performed using HPLC.

Results:

Two types of presentation of the results are given. First: specific growth characteristics are given in Tables IV.A and IV. B (biomass, seed yield, growth rate, GS activity, GPT activity, 2-oxoglutaramate activity, etc.). Second: photographs of offspring plants and their leaves are shown in comparison with plants with one transgene and wild-type plants and their leaves in FIG. 5 and FIG. 6, where it can be seen that plants with two transgenes show much larger sizes of the whole plant, much more large leaves and earlier and / or more abundant flowering compared to the parent plants with a single transgene or control wild-type plants.

As can be seen from Table IV.A, the offspring plants with two transgenes that make up these crosses showed a surprising increase in total biomass (raw tissue mass), with the raw tissue mass lying in the range of 45-89 grams per individual offspring plant, compared to 19- 24 grams per individual wild-type plant, that is, the average value of the increase is two or three-fold, and are highly productive, since there is an amazing four-fold increase in biomass compared to wild-type plants. Taking into account the values obtained for 24 progeny plants with two transgenes, the average biomass for one plant was about 2.75 higher than the average biomass for control plants of the wild type. Four lines of offspring showed an increase in the average weight of raw tissue per plant by about 2.5 times, while two lines showed a three-fold increase in the mass of raw tissue compared to wild-type plants.

Compared to generation lines with a single transgene, double-transgenic progeny plants showed more than additive growth enhancement. Whereas GPT lines with a single transgene showed a 50% increase in biomass compared to the wild type, and GS1 lines with a single transgene showed a 66% increase, progeny plants showed an increase of almost 200% compared to wild type plants.

Similarly, offspring plants carrying two transgenes bloomed earlier and more intensively than parent lines of the wild type and parent lines with one transgene, and much more seed bolls were formed, as well as seeds in general per plant. Taking into account the data in Table IV.A, on average, twice as many seed bolls formed in transgenic offspring compared to wild-type plants, moreover, in two of the highly productive plants, the number of seed bolls formed more than three times the number seed boxes in wild-type plants. The total seed yield of offspring plants, measured in relation to the mass of the plant, was two to four times higher than for wild-type plants.

Table IV.B shows the growth rate, biomass and yield and biochemical characteristics of line XX (line 3 self-pollinates further), compared with a single transgene line expressing GS1 and a wild-type control tobacco plant. All parameters improved significantly for transgenic plants carrying two transgenes (Line XX). It should be noted that 2-oxoglutaramate activity was almost 17 times higher, and the yield of seeds and deciduous biomass was three times higher for plants of line XX compared with control plants

TABLE IV.B Plant type Specific growth rate mg / g / d Deciduous biomass FWt, g fruits / flowers / buds Seed Harvest, g GS activity μmol / min / g FWt GPT Activity μmol / min / g FWt 2-oxo-glutaramate nmol / gFWt Analysis, transgenes Wild type, medium 228 21.40 28.5 1.02 7.75 16.9 68.9 no Line 1 GS 269 35.57 n.i. n.i. 11.6 n.i. 414 there is Line XX 339 59.71 62.9 2.94 16.3 243.9 1,153.6 there is n.i. - not measured

EXAMPLE 8: Obtaining double-transgenic pepper plants with two transgenes GS1 and GPT:

In this example, Big Jim chili pepper plants (cultivar New Mexico) transformed Arabidopsis GPT with the full length SEQ ID NO: 1 coding sequence under the control of the CMV 35S promoter and Arabidopsis GS1 coding sequence included in the SEQ ID NO: 6 under the control of the RuBisCo promoter using Agrobacterium-mediated transfer to seed boxes. After 3 days, the seeds were collected and used to transform T0 plants and screened for transformants. The resulting double-transgenic plants showed a higher number of bolls, higher growth rates and more significant biomass compared to control plants.

Materials and methods:

Pepper plants of Solanaceae Capisicum ("Big Jim" variety) transformed Arabidopsis GPT with the coding sequence of full length SEQ ID NO: 1 under the control of the CMV 35S promoter within the pMON expression vector (see Example 3), and Arabidopsis GS1 coding sequence included in SEQ ID NO: 6, under the control of the RuBisCo promoter within the pCambia 1201 expression vector (Tomato rubisco rbcS3C promoter: Kyozulka et al., 1993, Plant Physiol. 103: 991-1000; SEQ ID NO: 22; vector construction SEQ ID NO: 6) using Agrobacterium -mediated transfer to seed boxes.

In this and subsequent examples of Gambia 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 was equipped with a 35S CaMV promoter; this promoter was replaced by the RcbS-3C tomato promoter to control the expression of the target gene. Gambia 1201 vectors contain genes for a selectable marker of resistance to bacterial chlorphenicol and plant hygromycin. Gambia 1305.1 vectors contain genes for a selectable marker of resistance to bacterial chlorphenicol and hygromycin.

The transgenic expression vectors pMON (GPT transgene) and pCambia 1201 (GS transgene) were transferred to the isolated cultures of Agrobacterium tumefaciens strain LBA4404 using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9: 155-159).

Transformed agrobacteria were selected on a medium containing 50 μg / ml of either streptomycin for pMON constructs or chloramphenicol for Cambia constructs. Transformed agrobacterial cells were grown on LB culture medium containing 25 μg / ml antibiotic for 36 hours. At the end of the 36 hour growth period, the cells were harvested by centrifugation, and the cells for each transformation were resuspended in 100 ml of LB broth without antibiotic.

Pepper plants were then transformed with a mixture of the obtained agrobacterial cell suspensions using a transformation protocol in which the agrobacterium was injected directly into the seed chamber of developing pods. Briefly, 200 ml of the mixture was injected into developing capsules in order to inoculate immature seeds with an agrobacterium, essentially as described (Wang and Waterhouse, 1997, Plant Mol. Biol. Reporter 15: 209-215). In order to induce agrobacterial virulence and improve transformation efficiency, 10 μg / ml of acetosyringonone was added to the agrobacterial cultures before pod inoculation (see, Sheikholeslam and Weeks, 1986, Plant Mol. Biol. 8: 291-298).

Using a syringe, an amount of an agrobacterial vector mixture with a supply was injected into the pods and incubated for about 3 days. The seeds were then harvested and allowed to germinate, and developing plants were observed for their phenotypic characteristics, including growth and antibiotic resistance. Plants carrying transgenes were green, while non-transformed plants showed signs of chlorosis at the ends of the leaves. Strong growing transformants were then cultivated and compared to wild-type pepper plants grown under identical conditions.

Results:

The results are shown in FIG. 7 and Table V. FIG. 7 shows a GPT + GS photograph of a double transgenic pepper plant compared to a control plant grown over the same period under identical conditions. This photo shows a huge yield of pepper transgenic lines compared with the control plant.

Table V shows the biomass yield and GS activity, as well as the results of transgenic genotyping for the transgenic line, compared with the wild-type control. Based on the data in Table V, it is seen that the double transgenic progeny plants showed an incredible increase in total biomass (raw tissue mass), with the raw tissue mass ranging from 393 to 662 g per transgenic plant compared to the raw mass tissue an average of 328 g for a wild-type plant. The transgenic line A5 produces a total biomass of more than two times more than in control plants. Moreover, the yield of pepper plants of transgenic lines was very improved compared to wild-type plants, and they were 50% more than control plants (on average). It should be noted that one of the transgenic lines produced twice as many plants as the control plants on average.

EXAMPLE 9: Obtaining double-transgenic bean plants bearing ARABIDOPSIS GS1 and GPT transgenes:

In this example, yellow wax bean plants (Phaseolus vulgaris) transformed Arabidopsis GPT with the full length SEQ ID NO: 1 coding sequence under the control of the CMV 35S promoter within the pCambia 1201 expression vector, and Arabidopsis GS1 with the coding sequence included in SEQ ID NO: 6, under control of the RuBisCo promoter within the pCambia 1201 expression vector using agrobacterium-mediated flower transfer.

Materials and methods:

Transgenic expression vectors pCambia 1201-GPT (including construct SEQ ID NO: 27) and pCambia 1201-GS (including construct SEQ ID NO: 6) were transformed with isolated cultures of Agrobacterium tumefaciens strain LBA4404 using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9: 155-159). Transformed agrobacteria were selected on a medium containing 50 μg / ml chloramphenicol. Transformed agrobacterial cells were grown on LB culture medium containing 25 μg / ml antibiotic for 36 hours. At the end of the 36 hour growth period, the cells were harvested by centrifugation, and the cells from each transformation were resuspended in 100 ml of LB broth without antibiotic.

Bean plants were then transformed with a mixture of the obtained agrobacterial cell suspensions using a transformation protocol according to which the agrobacterium was injected directly into the flower structure (Yasseem, 2009, Plant Mol. Biol. Reporter 27: 20-28). In order to induce virulence of agrobacteria and improve transformation efficiency, 10 μg / ml of acetosyringonone was added to the cultures of agrobacteria before inoculation of the flower. Briefly, as soon as the flowers bloomed, the external structure encapsulating the reproductive organs was gently opened with tweezers in order to ensure the introduction of a mixture of agrobacteria, which was added to the flower structure, in an amount sufficient to fill the anther.

Plants were grown before the development of bean pods, and the seeds were harvested to generate transgenic plants. Transgenic plants were then grown together with control bean plants under identical conditions, photographed and phenotypic characteristics determined. Growth rates were measured for both transgenic plants and control plants. In this and other examples, glutamine synthetase (GS) activity was measured according to the methods of Shapiro and Stadtmann, 1970, Methods in Enzymology 17A: 910-922; and glutamine-phenylpyruvate transaminase (GPT) activity was evaluated according to the methods of Calderon et al., 1985, J. Bacteriol. 161: 807-809. See the details in Example 7 (Methods) above.

Results:

The results are presented in FIG. 8, FIG. 9 and in Table VI.

FIG. 8 shows data on the GPT + GS growth rate of the transgenic line A of the beans compared to control plants, including the height of the plants on different days during cultivation, as well as the number of buds, flowers, and pods. These data showed that GPT + GS twice transgenic bean plants are superior in growth to similar control plants. Transgenic plants grew taller, showed earlier flowering and produced more buds and flowers, and earlier developed pods and produced a greater number of pods compared to control plants of the wild type.

TABLE VI Transgenic Bean Plant Line A Plant type The output of the pods FWt, g GPT activity / nmol / h / g FWt GS activity μmol / min / rFWt Antibiotic resistance Wild type, average 126.6 101.9 25.2 Negative 2A 211.5 n.i. n.i. + 4A 207.7 n.i. n.i. + 5V 205.7 984.7 101.3 + WT - wild type; FWt is the mass of crude tissue; n.i. - not measured

Table VI shows the yield of bean pods, GPT and GS activity, as well as the antibiotic resistance status for transgenic lines compared to the wild-type control (average of several healthy control plants; control plants that did not grow well were excluded from the analysis). Based on the data in Table VI, double-transgenic progeny plants showed a significant increase in pod biomass (raw tissue mass) compared to control plants, with the pod biomass reaching more than 200 g per transgenic plant, compared to an average of 127 g wild-type plant, which represents an increase in the yield of pods of double-transgenic plants by more than 60%, compared with the control plant (s).

Finally, FIG. 9 shows a GPT + GS photograph of a double transgenic bean plant compared to a control plant grown for the same period of time under identical conditions, showing increased growth for the transgenic plant.

EXAMPLE 10: obtaining double transgenic bean plants bearing GS1 transgenes from ARABIDOPSIS and GPT from grapes:

In this example, yellow bean plants (Phaseolus vulgaris) were transformed with the full length grape GPT coding sequence included in SEQ ID NO: 8, under the control of the RuBisCo promoter inside the pCambia 1305.1 expression vector, and Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 , under the control of the RuBisCo promoter within the pCambia 1201 expression vector, using agrobacterium-mediated transfer into developing pods.

Materials and methods:

Transgenic expression vectors pCambia 1201-CPT (grape) (including construct SEQ ID NO: 8) and pCambia 1201-GS (including construct SEQ ID NO: 6) were transferred to isolated cultures of Agrobacterium tumefaciens strain LBA4404 using a standard electroporation method (McCormac et al ., 1998, Molecular Biotechnology 9: 155-159). Transformed agrobacteria were selected on a medium containing 50 μg / ml chloramphenicol. Transformed agrobacterial cells were grown on LB culture medium containing 25 μg / ml antibiotic for 36 hours. At the end of the 36 hour growth period, the cells were harvested by centrifugation, and the cells from each transformation were resuspended in 100 ml of LB broth without antibiotic.

Bean plants were then transformed with a mixture of the obtained agrobacterial cell suspensions using a transformation protocol according to which the agrobacterium was injected directly into the flower structure. In order to induce virulence of agrobacteria and improve the efficiency of transformation, 10 μg / ml of acetosyringonone was added to the cultures of agrobacteria before inoculation of the flower. Briefly, as soon as the flowers bloomed, the external structure encapsulating the reproductive organs was gently opened with tweezers in order to ensure the introduction of a mixture of agrobacteria, which was added to the flower structure, in an amount sufficient to fill the anther.

Plants were grown before the development of bean pods, and the seeds were harvested to generate transgenic plants. Transgenic plants were grown together with control bean plants under identical conditions, photographed and phenotypic characteristics were determined. Growth rates were determined both for transgenic plants and for control ones.

Results:

The results are shown in FIG. 10, FIG. 11 and in Table VII.

FIGURE 10 shows data on the growth rate of GPT + GS of the transgenic line G of bean plants, compared with control plants, where, in particular, data on the number of buds, flowers and pods are indicated. These data show that GPT + GS double transgenic plants are superior in growth to their wild-type counterparts. It should be noted that transgenic plants produce significantly more pods than control plants of the wild type.

TABLE VII Transgenic line G of bean plants: the output of pods Plant type The output of the bean pods FWt, g Antibiotic resistance Wild type, average 157.9 negative G1 200.5 + G2 178.3 + WT - wild type; FWt is the mass of crude tissue; n.i. - not measured

Table VII shows pod yield data and antibiotic resistance status for transgenic lines compared to wild-type control plants (average for several healthy control plants; control plants that did not grow well were excluded from the analysis). Based on the data given in Table VII, it is seen that double-transgenic progeny plants show a significantly increased biomass of bean pods (raw tissue mass) compared to control plants, and the yield of pod biomass is 200.5 g (line G1) and 178 g (line G2) per transgenic plant, compared with 158 grams, on average, per wild-type plant, which represents an increase in the yield of pods of double-transgenic plants by about 27%, compared to control plants.

Finally, FIG. 11 shows a photograph of GPT + GS of double transgenic bean plants compared to control plants grown over the same period under identical conditions. The transgenic plant shows a significant increase in size and biomass, larger leaves and more efficient flowering compared to the control plant.

EXAMPLE 11: obtaining double-transgenic wigna plants bearing ARABIDOPSIS GS1 and GPT transgenes:

In this example, normal Vigna plants transformed the Arabidopsis GPT coding sequence of full length SEQ ID NO: 1 under the control of the CMV 35S promoter inside the pMOM expression vector, and the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under the control of the RuBisCo promoter inside the expression vector pCambia 1201 using Agrobacterium-mediated flower transfer. Materials and methods correspond to Example 9, see above.

Results:

The results are presented in FIGS. 12 and 13, and in Table VI. FIG. 12 shows the relative growth rates for the GPT + GS of the Vigna transgenic line A and the wild-type Vigna control plant for some intervals during cultivation, including measuring (FIG. 12A) the height and length of the leaf, (FIG. 12B) trefoils and buds, and (FIG.12C) flowers, buds and pods. These data show that GPT + GS twice transgenic Vigna plants are superior in growth to their counterparts, which are control plants. Transgenic plants grew faster and were taller, had longer leaves and a higher rate of formation of pods and flowers, compared to control plants of the wild type.

TABLE VIII Transgenic line A Wigny Plant type The output of pods, FWt, g GPT activity mol / h / gFWt GS activity μmol / min / gFWt Antibiotic resistance Wild type, average 74.7 44.4 28.3 Negative 4A 112.8 NM 41.3 + 8B 113.8 736.2 54.9 + WT - wild type; FWt is the mass of crude tissue; n.i. - not measured

Table VIII shows the yield of pods, GPT and GS activity, as well as the antibiotic resistance status for transgenic lines compared to the wild-type control (average of several healthy control plants; control plants that did not grow well were excluded from the analysis). According to the data in Table VIII, it can be seen that double-transgenic plants showed a significant increase in pod biomass (mass of raw pod tissue) compared to control plants, and the average yield of pod biomass for transgenic plants was about 52% higher compared to the yields measured for control plant (s).

Finally, FIG. 13 shows a GPT + GS photograph of double-transgenic bean plants compared to control plants grown over the same period under identical conditions, showing increased biomass and yield of pods for transgenic plants compared to control wild-type plants.

EXAMPLE 12: obtaining double transgenic plants Vigna carrying GS1 transgenes from ARABIDOPSIS and GPT from grapes:

In this example, normal Vigna plants were transformed with the coding sequence included in SEQ ID NO: 8, the full length GPT of the grape under the control of the RuBisCo promoter inside the pCambia 1305.1 expression vector (including the construct SEQ ID NO: 8), and Arabidopsis GS1 coding sequence included in SEQ ID NO: 6, under the control of the RuBisCo promoter within the pCambia 1201 expression vector (including construct SEQ ID NO: 6) using agrobacterium-mediated flower transfer. The materials and methods are consistent with Example Example 11, see above.

Results:

The results are presented in FIG. 14 and 15, and in Table IX.

FIG. 14 shows the relative growth rates for GPT + GS of the Vigna transgenic G line and the wild type Vigna control plant. These data show that the transgenic plants above (FIG.14A) produce a significantly larger number of flowers, buds and pods (FIG.14B), and more quickly develop trefoils and leaf buds (FIG.14C).

TABLE IX GS Wigny Transgenic Line Plant type The output of pods, FWt, g GPT activity nmol / h / g FWT GS activity μmol / min / gFWt Antibiotic resistance Wild type, average 59.7 44.4 26.7 Negative G9 102.0 555.6 34.5 + WT - wild type; FWt is the mass of crude tissue; n.i. - not measured

Table IX shows the yield of pods, GPT and GS activity, as well as the antibiotic resistance status for transgenic lines compared to the wild-type control (average of several healthy control plants; control plants that did not grow well were excluded from the analysis). According to the data in Table IX, it is clear that double-transgenic plants showed a significant increase in pod biomass (mass of raw pod tissue) compared to control plants, and the average yield of pod biomass for transgenic plants was about 70% higher compared to yields. measured for the control plant (s).

Finally, FIG. 15 shows a GPT + GS photograph of double transgenic bean plants, compared to control plants grown over the same period under identical conditions, showing increased biomass and leaf size for transgenic plants, compared to wild type control plants.

EXAMPLE 13: obtaining double-transgenic plants ALFALFA carrying transgenes ARABIDOPSIS GS1 and GPT:

In this example, normal Alfalfa plants (Medicago sativa, var Ladak) transformed Arabidopsis GPT with the full length CMQ sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3 above), and Arabidopsis GS1 with the coding sequence included in SEQ ID NO: 6, under the control of the RuBisCo promoter within the pCambia 1201 expression vector (including construct SEQ ID NO: 6) using agrobacterium-mediated transfer to seedlings. Agrobacterial vectors and mixtures were obtained for inoculation of germinating plants, as described in Example 11, see above.

Inoculation of germinating plants:

When Alfalfa sprouting plants were less than about 1/2 inch in length, they were wrapped in a paper towel that was saturated with a mixture of agrobacteria containing both transgenic constructs. Germinating plants were left in paper towels for two to three days, removed and planted in the soil. The resulting maintenance and control plants were then grown for the first 30 days in a cultivation chamber, then cultivated in a greenhouse and then harvested 42 days after germination. At this point, flowers were observed only in transgenic plants, since only immature buds were observed in wild-type plants. Plants were characterized by flowering and total biomass.

Results:

The results are presented in Table X. The data show that transgenic Alfalfa plants grow faster, bloom earlier, and biomass yield is on average about 62% higher compared to control plants.

TABLE X Transgenic ALFALFA Compared to Controls Plant type The biomass during the collection, g Flowering stage Lightly marked kidneys Wild type, average 6.03 No swelling of the kidneys. No bloom Transgene No. 5 10.38 4 open flowers Transgene No. 11 9.03 Flower bud swelling Transgene No. 13 9.95 Flower bud swelling

EXAMPLE 14: obtaining double-transgenic Cantaloupe plants carrying the transgenes ARABIDOPSIS GS1 and GPT:

In this example, Cantaloupe plants (Cucumis melo var common) transformed Arabidopsis GPT with the coding sequence of SEQ ID NO: 1 full length under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3, above), and Arabidopsis GS1 with the coding sequence included in SEQ ID NO: 6, under the control of the RuBisCo promoter within the pCambia 1201 expression vector (including construct SEQ ID NO: 6) using agrobacterium-mediated transfer by injection into developing melon fruits. Agrobacterial vectors and mixtures were obtained by inoculation of melon fruits, as described in Example 8 above. Inoculation of developing melon fruits was carried out as described in Example 8. Plants were characterized by flowering status and total biomass, compared to control wild-type plants grown under identical conditions.

The results are presented in FIG.16 and in Table XI. According to the data in Table XI, transgenic plants show a significant increase in the biomass of plant leaves, compared with control plants, with an average increase of biomass of 63%. Moreover, there was a significant increase in the yield of flowers and buds for all five transgenic lines. In control plants, on average, no flowers and only 5 buds were observed. In contrast, transgenic plants observed from 2 to 5 flowers per plant, and from 21 to 30 buds per plant, indicating a significantly higher growth rate and higher yield of flowers. An increased yield of flowers, as one would expect, is associated with a corresponding higher yield of melon fruits for transgenic plants. Based on FIG.16 (photo comparison of transgenic plants of Cantaloupe with control plants of Cantaloupe), transgenic plants of Cantaloupe show a tremendous increase in height, total biomass and flowering status compared to control plants.

TABLE XI Cantaloupe transgenic plants compared to controls Plant type Biomass sheets FWt, g Flowers / buds at the time of collection Antibiotic resistance Wild type, average 22.8 0/5 negative Line 1 37.0 3/21 + Line 2 35.0 2/30 + Line 3 37.1 3/27 + Line 4 40.6 5/26 + Line 5 35.7 4/30 + FWt - wet tissue mass

EXAMPLE 15: obtaining double-transgenic plants of pumpkin ordinary, bearing transgenes ARABIDOPSIS GS1 and GPT:

In this example, common squash plants (Cucurbita maxima) transformed Arabidopsis GPT with the full length CMQ 35S promoter coding sequence SEQ ID NO: 1 inside the pMON316 expression vector (see Example 3 above), and Arabidopsis GS1 with the coding sequence included in SEQ ID NO: 6, under the control of the RuBisCo promoter within the pCambia 1201 expression vector (including construct SEQ ID NO: 6), using agrobacterium-mediated transfer by injection into developing pumpkin fruits, essentially as described in Example 14 above. Transgenic and control plants of common squash were grown under identical conditions until buds appeared in control plants, then all plants were characterized by flowering status and total biomass.

The results are presented in FIG.17 and in Table XII. Based on the data in Table XII, it can be concluded that transgenic plants show a significant increase in the biomass of the leaves of the plants compared to control plants, with an average biomass yield of 67% higher than control plants. Moreover, an increase in flower yield was observed in four of the five transgenic lines compared to the control. In control plants, about 4 buds at sacrifice were observed (on average). In contrast, in four transgenic plant lines, 8 to 15 buds per plant were observed, which represents an increase in yield from about two to about four times.

TABLE XII Comparison of transgenic pumpkin plants with control Plant type Leaf biomass FWt, g Flowers Antibiotic resistance Wild type, average 47.7 4.2 Negative Line 1 (Photo) 82.3 8 Line 2 74.3 8 + Line 3 80.3 9 + Line 4 (Photo) 77.8 four + Line 5 84.5 fifteen + FWt - wet tissue mass

FIGURE 17 (photograph of a comparison of transgenic pumpkin plants with control plants) shows that transgenic pumpkin plants show a significant increase in plant size, total biomass, number and size of leaves, compared to control plants.

EXAMPLE 16: obtaining double-transgenic plants ARABIDOPSIS carrying transgenes ARABIDOPSIS GS1 and GPT:

In this example, Arabidopsis thaliana plants were transformed with a truncated sequence encoding Arabidopsis GPT, SEQ ID NO: 18, under the control of the CMV 35S promoter within the pMON316 expression vector (see Example 3 above), and after that, transgenic plants transformed Arabidopsis GS1 with the coding sequence included in SEQ ID NO: 6, under the control of the RuBisCo promoter within the pCambia 1201 expression vector (including the 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). Acrobacterial vectors pMON316 carrying GPT and pCambia 1201 carrying GS1 were obtained as described in Examples 3 and 11, respectively.

Transformation of two different cultures of agrobacteria with either pMon 316 + Arabidopsis GTP construct or Gambia 1201 + Arabidopsis GS construct was performed by electroporation using the method of Weigel and Glazebrook 2002. The transformed agrobacteria were then grown with antibiotic selection, harvested by centrifugation, resuspended in LB broth with antibiotic and was used in the implementation of the method of "floral dip" in the inflorescence of Arabidopsis. The Arabidopsis plants obtained by the Floral dip methods were grown to maturity, allowed them to self-pollinate, and seeds were harvested. Seeds from two plants treated with the Floral dip method were first grown on a medium containing 20 μg / ml kanamycin, and using the following conventional selection procedures, surviving seedlings were transferred to a medium containing 20 μg hygromycin. The plants (3) that survived the selection process for the two antibiotics self-pollinated and the seeds were collected. T1 generation seeds sprouted on MS medium containing 20 μg / ml hygromycin, and the surviving seedlings were allowed to grow to a mature plant state, self-dust, and seeds were collected. T2 generation seeds were used to study subsequent growth.

The results are presented in FIG. 18 and in Table XIII. According to Table XIII, which presents data for six transgenic Arabidopsis plants (averaged), transgenic plants show elevated levels of activity of both GPT and GS. GPT activity was twenty times higher compared to control plants. Moreover, the raw tissue mass of transgenic plants was also four times higher on average compared to control plants (average). A photograph of young transgenic Arabidopsis plants, compared to control wild type Arabidopsis plants grown under identical conditions, is shown in FIG. 18 and shows a uniform and very significant increase in growth / biomass for transgenic plants compared to control plants.

TABLE XIII transgenic plants ARABIDOPSIS compared to control Plant type Leaf biomass FWt, g GPT activity nmol / h / g FWt GS activity μmol / min / gFWt Transgene Analysis Wild type, average 0.246 18.4 7.0 Negative transgene 1.106 395.6 18.2 Positive

EXAMPLE 17: obtaining transgenic tomato plants bearing ARABIDOPSIS GPT and GS1 transgenes:

In this example, tomato plants (Solanum lycopersicon, cultivar "Money Maker") were transformed with Arabidopsis GPT coding sequence SEQ ID NO: 1 full length under the control of the CMV 35S promoter within the expression vector pMON316 (see Example 3 above), and Arabidopsis GS1 coding sequence, included in SEQ ID NO: 6, under the control of the RuBisCo promoter within the pCambia 1201 expression vector (including construct SEQ ID NO: 6). Transgenic single transgenic tomato plants (GPTs) were generated and grown before flowering, essentially as described in Example 4. The Arabidopsis GS1 transgene was then introduced into T0 single transgenic plants using agrobacterium-mediated transfer by injection directly into flowers (as described in Example 8). Transgenic and control tomato plants were grown under identical conditions and characterized by the characteristics of the growth phenotype. The obtained T0 twice transgenic plants were then grown to maturity, photographed with control tomato plants, and phenotypic characteristics were determined.

The results are presented in FIG.19 and in Table XIX. From Table XIX it is seen that twice transgenic tomato plants show a significant increase in the biomass of plant leaves compared to control plants, with an average increase in biomass yield of 45% compared to control plants. Moreover, there was also a 70% increase in tomato yield for transgenic lines compared to control plants (for example, 51 tomatoes were collected from line 4C, on the contrary, on average about 30 tomatoes were collected from control plants on average). Transgenic plants showed a much higher level of GPT activity (for example, line 4C showed about a 32-fold increase in GPT activity compared to GPT activity, measured on average for control plants). GS activity was also higher in transgenic plants compared with the control ones (almost twice for the 4C line).

As for the growth phenotype, as can be seen in FIG. 19, leaves of a significantly larger size develop in transgenic tomato plants compared to control plants (FIG. 19A). In addition, you can see that the transgenic tomato plants are significantly larger, higher and have a larger total biomass (see FIG.19B).

TABLE XIX growth and reproduction of transgenic tomato plants Plant type Leaf biomass FWt, g The number of harvested tomatoes GPT activity nmol / h / g FWt GS activity μmol / min / gFWt Transgene Analysis Wild type, average 891 30.2 287 14.27 Negative Line 6C 1288 43 9181 18.3 + Line 4C 1146 51 1718 26.4 +

EXAMPLE 18: obtaining transgenic plants CAMILENA bearing the transgenes ARABIDOPSIS GPT and GS1:

In this example, Camelina plants (Camelina sativa, Var MT 303) transformed Arabidopsis GPT with the full length SEQ ID NO: 1 coding sequence under the control of the RuBisCo promoter within the pCambia 1201 expression vector, and Arabidopsis GS1 with the coding sequence included in SEQ ID NO: 6, under control of the RuBisCo promoter within the pCambia 1201 expression vector using Agrobacterium-mediated transfer to germinating seeds according to the method described in Chee et al., 1989, Plant Physiol. 91: 1212-1218. Agrobacterial vectors and mixtures were obtained for inoculation of seeds, as described in Example 11 above.

Camelina transgenic and control plants were grown under identical conditions (30 days in a seed germination chamber and then transferred to a greenhouse for cultivation) for 39 days, and were characterized by biomass, growth characteristics and flowering stage.

The results are presented in Table XX and FIG. 20. According to Table XX, it can be seen that the total biomass in transgenic plants, on average, is almost twice the biomass of control plants. The diameter of the canopy also increased significantly in transgenic plants. FIGURE 20 shows a photograph of the transgenic Camelina plants in comparison with the control. Transgenic plants are noticeably larger and show improved flowering.

TABLE XX CAMELINA transgenic plants compared to control Plant type Canopy Height / Diameter Inches Biomass, g flowering stage Wild type, average 14/4 8.35 partial flowering transgene C-1 15.5 / 5 16.54 full bloom transgene C-3 14/7 14.80 initial flowering

EXAMPLE 19: GPT Transgenic Activity of Barley in Plants

In this example, the putative barley GPT coding sequence was isolated and expressed from a transgenic construct using an in planta transient expression assay. The biologically active recombinant barley GPT was produced and catalyzed increased synthesis of 2-oxoglutaramate, as confirmed by HPLC. The coding sequence of the GPT of barley (Hordeum vulgare) was determined and synthesized. The DNA sequence of the barley GPT coding sequence used in this example corresponds to SEQ ID NO: 14, and the amino acid sequence of the GPT encoded protein is SEQ ID NO: 15.

The barley GPT coding sequence was inserted into cambia vector 1305.1, and transferred to LBA404 strain Agrobacterium tumefaciens using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9: 155-159), then placed on LB plates containing hygromycin (50 μg / ml). Antibiotic resistance colonies of agrobacteria were selected for analysis.

Analysis of the transient expression of tobacco leaves consisted of injecting a suspension of transformed agrobacteria (1.5-2.0 OD 650) into fast-growing tobacco leaves. Intradermal injections were made into a mesh on the surface of the sheet to expose a significant surface of the sheet to agrobacteria. Then the plants grew for 3-5 days, after which the tissue was extracted as described for all other tissue extracts, and GPT activity was measured.

The GPT activity in inoculated leaf tissue (1217 nanomol / gFWt / h) was three times higher than the GPT activity measured in leaf tissues of control plants (407 nanomol / gFWt / h), which indicates that the Hordeum GPT construct controls the expression of biologically active GPT in a transgenic plant.

EXAMPLE 20: Isolation and expression of the coding sequence of the recombinant rice GPT gene and analysis of biological activity

In this example, the putative rice GPT coding sequence was isolated and expressed in E. coli. Biologically active recombinant rice GPT was produced and catalyzed increased synthesis of 2-oxoglutaramate, as confirmed by HPLC.

Materials and methods:

Rice GPT coding sequence and expression in E. coli:

The coding sequence of GPT rice (Oryza sativa) was determined and synthesized, inserted into a PET28 vector and expressed in E. coli. Briefly, E. coli cells were transformed with an expression vector, and transformants were grown overnight in LB broth diluted to an optical density (OD) of 0.4, expression was induced with isopropyl-B-D-thiogalactoside (0.4-micromolar), grown for 3 hours and collected . A total of 25 X 10 ⁶ cells were analyzed for biological activity using NMR analysis below. Non-transformed wild-type E. coli cells were analyzed as a control. As an additional control, E. coli cells transformed with an empty vector were used.

The DNA sequence of the rice GPT coding sequence used in this example corresponds to SEQ ID NO: 10, and the encoded amino acid sequence of the GPT protein corresponds to SEQ ID NO: 11.

HPLC analysis of 2-oxoglutaramate:

HPLC was used to determine the production of 2-oxoglutaramate by GPT overexpressing E. coli cells, following a modification of Calderon et al., 1985, J Bacteriol 161 (2): 807-809. Briefly, a modified extraction buffer was used containing 25 mM Tris-HCl pH 8.5, 1 mM EDTA, 20 μM pyridoxalphosphate, 10 mM Cysteine and about 1.5% (v / v) mercaptoethanol. Samples (E. coli cell lysate, 25 X 10 ⁶ cells) were added to the extraction buffer at a ratio of about 1/3 (w / v), incubated for 30 minutes at 37 ° C, and stopped using 200 μl of 20% TCA After about 5 minutes, the analyzed mixture was centrifuged, and the supernatant was used to quantify 2-oxoglutaramate by HPLC using an ION-300 7.8 mm ID X 30 cm L column, with a mobile phase of 0.01 N H ₂ SO ₄ , flow rate of about 0.2 ml / min at 40 ° C. The injection volume was about 20 μl, and the retention time was 38 to 39 minutes. Detection was carried out using UV radiation with a wavelength of 210 nm.

Comparison by NMR analysis with authentic 2-oxoglutaramate was used to confirm that the full length Arabidopisis sequence expresses GPT with 2-oxoglutaramate synthesis activity. Briefly, authentic 2-oxoglutaramate (a structure confirmed by NMR) was obtained by chemical synthesis to confirm the results of HPLC analysis above by confirming that the product of analysis (a molecule synthesized in response to expressed GPT) and authentic 2-oxoglutaramate elute at the same retention time. In addition, when mixed together, the assay product and authentic compound elute with the appearance of a single peak. In addition, confirmation of the results of the HPLC analysis also includes monitoring the disappearance of the glutamine substrate, while it was shown that there is a 1: 1 molar stoichiometry between the glutamine consumption and the 2-oxoglutaramate produced. The analysis procedure always includes two controls, one without enzyme and one without glutamine. The first shows that the production of 2-oxoglutaramate depends on the presence of the enzyme, and the second shows that the production of 2-oxoglutaramate depends on the substrate of glutamine.

Results:

Expression of the GPT coding sequence of rice SEQ ID NO: 10 resulted in overexpression of a recombinant GPT protein having bioactivity associated with catalysis of the synthesis of 2-oxoglutaramate. In particular, 1.72 nanomoles of 2-oxoglutaramate was observed in E. coli cells overexpressing recombinant rice GPT, compared with only 0.02 nanomoles of 2-oxoglutaramate in control E. coli cells, i.e. an 86-fold increase in the level of activity compared to the control.

EXAMPLE 21: Isolation and expression of the coding sequence of the recombinant soybean GPT gene and analysis of biological activity

In this example, the putative soybean GPT coding sequence was isolated and expressed in E. coli. Biologically active recombinant soybean GPT was produced and catalyzed increased synthesis of 2-oxoglutaramate, as confirmed by HPLC.

Materials and methods:

Soybean GPT coding sequence and expression in E. coli:

The soybean GPT coding sequence (Glycine max) was determined and synthesized, inserted into a PET28 vector, and expressed in E. coli. Briefly, E. coli cells were transformed with an expression vector, and transformants were grown overnight in LB broth diluted to an optical density (OD) of 0.4, expression was induced with isopropyl-B-D-thiogalactoside (0.4-micromolar), grown for 3 hours and collected . A total of 25 X 10 ⁶ cells were analyzed for biological activity using HPLC analysis below. Non-transformed wild-type E. coli cells were analyzed as a control. As an additional control, E. coli cells transformed with an empty vector were used.

The DNA sequence of the soybean GPT coding sequence used in this example corresponds to SEQ ID NO: 12, and the encoded amino acid sequence of the GPT protein corresponds to SEQ ID NO: 13.

HPLC analysis of 2-oxoglutaramate:

HPLC was used to determine the production of 2-oxoglutaramate by GPT overexpressing E. coli cells as described in Example 20 above.

Results:

Expression of the soybean GPT coding sequence of SEQ ID NO: 12 resulted in overexpression of a recombinant GPT protein having bioactivity associated with catalysis of the synthesis of 2-oxoglutaramate. In particular, 31.9 nanomoles of 2-oxoglutaramate was observed in E. coli cells overexpressing recombinant soybean GPT, compared with only 0.02 nanomoles of 2-oxoglutaramate in control E. coli cells, i.e. an almost 1600-fold increase in the level of activity compared to the control.

EXAMPLE 22: Isolation and Expression of the Coding Sequence of the Recombinant Striped Percina GPT Gene and Biological Activity Analysis

In this example, the putative striped percine GPT coding sequence was isolated and expressed in E. coli. The biologically active recombinant stripe-perine GPT was produced and catalyzed an increased synthesis of 2-oxoglutaramate, as confirmed by HPLC.

Materials and methods:

The coding sequence of GPT striped percina and expression in E. coli:

The GPT coding sequence of striped percina (Danio rerio) was determined and synthesized, inserted into a PET28 vector and expressed in E. coli. Briefly, E. coli cells were transformed with an expression vector, and transformants were grown overnight in LB broth diluted to an optical density (OD) of 0.4, expression was induced with isopropyl-BD-thiogalactoside (0.4-micromolar), grown for 3 hours and collected. A total of 25 X 10 ⁶ cells were analyzed for biological activity using HPLC analysis below. Non-transformed wild-type E. coli cells were analyzed as a control. As an additional control, E. coli cells transformed with an empty vector were used.

The DNA sequence of the striped pepperine GPT coding sequence used in this example corresponds to SEQ ID NO: 16, and the encoded amino acid sequence of the GPT protein corresponds to SEQ ID NO: 17.

HPLC analysis of 2-oxoglutaramate:

HPLC was used to determine the production of 2-oxoglutaramate by GPT overexpressing E. coli cells as described in Example 20 above.

Results:

Expression of the GPT coding sequence of stripe pepper SEQ ID NO: 12 resulted in overexpression of a recombinant GPT protein having bioactivity associated with catalysis of the synthesis of 2-oxoglutaramate. In particular, 28.6 nanomoles of 2-oxoglutaramate was observed in E. coli cells overexpressing recombinant striped pepper perch GPT, compared with only 0.02 nanomoles of 2-oxoglutaramate in control E. coli cells, i.e. a more than 1400-fold increase in activity level compared to control.

EXAMPLE 23: Obtaining and expression of the coding sequence of the recombinant truncated GPT gene ARABIDOPSIS and analysis of biological activity

In this example, two different truncated Arabidopsis GPT coding sequences were obtained and expressed in E. coli in order to evaluate the activity of GPT proteins in which the putative choroplastic signal peptide is missing or truncated. Recombinant GPT truncated proteins corresponding to the Arabidopsis GPT full length amino acid sequence SEQ ID NO: 2, truncated with the removal of the first 30 amino terminal amino acid residues, or the first 45 amino terminal amino acid residues, were successfully expressed and showed increased activity catalysis 2 -oxoglutaramate, as confirmed by HPLC.

Materials and methods:

Truncated Arabidopsis GPT coding sequences and expression in in E. coli:

The DNA coding sequence for truncating Arabidopsis thaliana GPT coding sequence SEQ ID NO: 1 was determined, 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 corresponds to SEQ ID NO: 20 (-45 AA construct), and the corresponding truncated amino acid sequence of the GPT protein corresponds to SEQ ID NO: 21. Briefly, E. coli cells were transformed with an expression vector, and transformants were grown overnight in LB broth diluted to an optical density of 0.4, expression was induced with isopropyl-B-D-thiogalactoside (0.4-micromolar), grown for three hours and harvested. A total of 25 X 10 ⁶ cells were analyzed for biological activity using HPLC as described in Example 20. Non-transformed E. coli wild-type cells were analyzed as a control. As an additional control, E. coli cells transformed with an empty vector were used.

The expression of the truncated -45 Arabidopsis GPT coding sequence of SEQ ID NO: 20 resulted in overexpression of the biologically active recombinant protein GPT (biological activity of the catalysis of the synthesis of 2-oxoglutaramate). In particular, 16.1 nanomoles of 2-oxoglutaramate was observed in E. coli cells overexpressing truncated -45 GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate in control E. coli cells, i.e. a more than 800-fold increase in activity level compared to control. For comparison, the full-length Arabidopsis gene coding sequence expressed in the same E. coli cells showed 2.8 nanomoles of 2-oxoglutaramate, or roughly less than one fifth of the activity observed for the truncated recombinant GPT protein.

EXAMPLE 24: Germination of GPT + GS TOBACCO SEEDS. TOLERANCE FOR HIGH SALT CONCENTRATIONS

In this example, the seeds of the double-transgenic tobacco line XX-3 (Cross 3 in Table 4, see Example 7) were tested by examining seed germination in order to assess tolerance to high salt concentrations.

Materials and methods:

The tobacco seeds of wild-type plants and XX-3 populations were sterilized (5% bleach solution for 5 minutes, and then washed with 10% ethanol for 3 minutes) and washed with sterile distilled water. The sterilized seeds were then placed on Murashige-Skoog medium (10% agarose) without sucrose, and containing either 0 or 200 mM NaCl. Seeds grew in the dark for two days, and then 6 days with a photoperiod of 16: 8 at 24 ° C. On the eighth day, the germination rate was determined by measuring the percentage of seeds relative to the control or transgenic plants that sprouted.

Results:

The results are presented in Table XXI below. The germination rate for the seeds of the transgenic plant line, at zero salt content, was the same as for the seeds of the wild-type control plants. On the contrary, the germination rate for seeds of the transgenic plant line at a very high salt content was much higher than the germination rate for the wild-type control seeds. While more than 81% of the seeds of transgenic plants germinated under conditions of high salt content, only about 9% of the seeds of control wild-type plants germinated over the same period of time. These data show that transgenic seeds can germinate very well at high salt concentrations, which is an important feature when plants grow in areas with increasing salt concentration in water and / or soil.

TABLE XXI Germination of transgenic tobacco plants and their tolerance to high salt content Plant type Control (0 mM NaCl) Test (200 mM NaCl) % Germination % Germination Wild type 92, 87, 94 9, 11, 8 Transgenic line XX-3 92, 91, 94 84, 82, 78

All publications, patents, and patent applications cited herein are incorporated herein by reference, as if it was expressly indicated that each individual publication or patent application was incorporated by reference.

The scope of the present invention is not limited to the embodiments of the present invention presented herein, which are intended only to illustrate individual objects of the present invention, and which find functional equivalents within the scope of the present invention. Various modifications to the options and methods of the present invention, in addition to those described herein, will be apparent to those skilled in the art based on the foregoing description and knowledge of the art, and therefore are also within the scope of the present invention. Such modifications or other embodiments of the present invention may be practiced without departing from the spirit and scope of the present invention.

Sequence table:

SEQ ID NO: 1 Arabidopsis glutamine-phenylpyruvate-transaminase DNA coding sequence:

SEQ ID NO: 2 Arabidopsis GPT amino acid sequence

SEQ ID NO: 3 Alfalfa GS1 DNA coding sequence (upper case) with 5 'and 3' untranslated sequences (shown in lower case).

SEQ ID NO: 4 Alfalfa GS1 amino acid sequence

SEQ ID NO: 5 Alfalfa GS1 DNA coding sequence (capital letters) with 5 'and 3' untranslated sequences (shown in lowercase letters) and vector sequences from Clal to Smal / Sspl and from Sspl / Smal to Sall / Xhol (lowercase, underlined )

SEQ ID NO: 6 Arabidopsis GS1 coding sequence Gambia 1201 vector + rbcSSC + arabidopsis GS1 (bold ATG is the initiation site),

SEQ ID NO: 7 Arabidopsis GS1 Amino Acid Sequence Vector sequences at the N-ends are shown in italics

SEQ ID NO: 8 DNA sequence encoding GPT of grapes Gambia 1305.1 with (3 'end) rbcS3C + Vitis vinifera GPT (grape) is shown. Bold ATG represents the initiation site, parentheses indicate the catl intron, and underlined actagt is the spel cloning site used for splicing into the GPT gene.

SEQ ID NO: 9 Amino Acid Sequence of GPT

SEQ ID NO: 10 DNA coding sequence for rice GPT GPT rice codon optimized for expression in E. coli; untranslated sequences are shown in lower case

SEQ ID NO: 11 The amino acid sequence of rice GPT includes the amino terminal amino acids MW for cloning and His tag sequences from the pet28 vector, shown in italics.

SEQ ID NO: 12 DNA coding sequence of GPT soybean TOPO 151D WITH SOYBEAN for expression in E. coli

From the start codon. Vector sequences in italics

SEQ ID NO: 13 amino acid sequence of soybean GPT. Translated protein product, vector sequences are shown in italics.

SEQ ID NO: 14 DNA coding sequence for GPT barley. The coding sequence from the remote intron initiation site

SEQ ID NO: 15 amino acid sequence of the barley GPT. Translated sequence from initiation site (remote intron)

SEQ ID NO: 16 DNA coding sequence for the GPT striped perine Danio rerio sequence for expression in E. coli. The nucleotides in bold and italics are added for cloning to or from the pET28b vector.

SEQ ID NO: 17 amino acid sequence of GPT of striped pepper Amino acid sequence of Danio rerio cloned and expressed in E. coli (amino acids in bold and italics are added from the vector / for cloning and His tag at the C-terminus)

SEQ ID NO: 18 DNA sequence of the truncated Arabidopsis GPT -30 construct Arabidopsis GPT coding sequence with 30 amino acids deleted from the target sequence.

SEQ ID NO: 19 amino acid sequence of truncated Arabidopsis GPT -30 construct

SEQ ID NO: 20: DNA sequence of the truncated Arabidopsis GPT -45 construct Arabidopsis GPT coding sequence with 45 deleted residues in the target sequence

SEQ ID NO: 21: Amino Acid Sequence of Truncated Arabidopsis GPT -45 Construction

SEQ ID NO: 22: tomato promoter Rubisco TOMATO RuBisCo rbcSSC promoter sequence from Kpnl to Ncol

SEQ ID NO: 23: DNA coding sequence for GPT bamboo

SEQ ID NO: 24: The amino acid sequence of GPT bamboo

SEQ ID NO: 25: 1305.1 + rbcS3C promoter + catl intron with the GPT rice gene.

Cambia 1305.1 with (3 'end) rbcS3C + GPT coding sequence of rice. An underlined ATG is an initiation site, parentheses indicate a catl intron, and an underlined actagt is a spel cloning site used for splicing into a rice gene.

SEQ ID NO: 26: SEQUENCE OF GPT OF BARLEY IN VECTOR Cambia 1305.1 with (3 'end) rbcS3C + hordeum IDI4 coding sequence. An underlined ATG is an initiation site, parentheses indicate a catl intron, and an underlined actagt is a spel cloning site used for splicing into the hordeum gene.

SEQ ID NO: 27 Gambia 1201 + Arabidopsis GPT coding sequence (35S CaMV promoter shown in italics)

SEQ ID NO: 28 Gambia p1305.1 with (3 'end) rbcS3C + Arabidopsis GPT coding sequence. An underlined ATG is an initiation site, parentheses indicate a catl intron, and an underlined actagt is a cloning site used for splicing into the Arabidopsis gene.

SEQ ID NO: 29 Arabidpsis GPT coding sequence (mature protein, non-target sequence)

SEQ ID NO: 30 Arabidpsis GPT amino acid sequence (mature protein, non-target sequence)

SEQ ID NO: 31 amino acid sequence of grape GPT (mature protein, non-target sequence)

SEQ ID NO: 32 Rice GPT amino acid sequence (mature protein, non-target sequence)

SEQ ID NO: 33 amino acid sequence of soybean GPT (-1 mature protein, non-target sequence)

SEQ ID NO: 34 amino acid sequence of barley GPT (mature protein, non-target sequence)

SEQ ID NO: 35 Amino Acid Sequence of GPT Striped Percina (Mature Protein, Non-Target Sequence)

SEQ ID NO: 36 amino acid sequence of GPT bamboo (mature protein, non-target sequence)

Claims (40)

1. A transgenic plant containing a transgene of glutamine-phenylpyruvate transaminase (GPT) and a transgene of glutamine synthetase (GS), where each GPT transgene and GS transgene is operably linked to a plant promoter,
where the transgenic plant exhibits increased biomass compared to the wild-type analogue or untransformed plant;
where the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, and
where the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4 and SEQ ID NO: 7. 2. The transgenic plant according to claim 1, where the GS transgene is a GS1 transgene. 3. The transgenic plant according to claim 1 or 2, where the GPT and GS transgenes are integrated into the plant genome. 4. The transgenic plant according to claim 3, wherein the transgenic plant is a monocotyledonous plant. 5. The transgenic plant according to claim 3, where the transgenic plant is a dicotyledonous plant. 6. A transgenic seed for producing a plant having an increased biomass containing a glutamine phenyl pyruvate transaminase (GPT) transgene and a glutamine synthetase transgene (GS), where each GPT transgene and GS transgene is operably linked to a plant promoter,
where a plant grown from a transgenic seed exhibits an increased biomass yield compared to a wild-type analogue or untransformed plant;
where the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, and
where the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4 and SEQ ID NO: 7. 7. A method of producing transgenic plants having increased production of 2-oxoglutaramate by transforming plants with transgenes that encode 2-oxoglutamate synthesis pathway enzymes to thereby increase the biomass of transgenic plants compared to a wild-type analog or untransformed plant, comprising:
(a) introducing a transgene of glutamine-phenylpyruvate transaminase (GPT) into plants;
(b) introducing a glutamine synthetase transgene (GS) into plants;
(c) expression of the GPT transgene and GS transgene in transgenic plants; and
(d) selecting a transgenic plant having an increased biomass compared to a plant of the same species that does not contain a GPT transgene or a GS transgene, where said transgenic plant produces more than 2-oxoglutamate relative to a plant of the same species that does not contain a GPT transgene or a GS transgene ,
where the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, and
where the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4 and SEQ ID NO: 7. 8. The method according to claim 7, where the transgenic plant contains at least one additional growth characteristic selected from the group consisting of earlier flowering, earlier budding, increased plant height, enhanced flowering, enhanced budding, larger leaf size, increased yield fruits or pods and increased seed yield. 9. The method of claim 7, further comprising collecting seeds from a transgenic plant and selecting seeds that exhibit improved germination under very high salt conditions. 10. The method of claim 9, further comprising growing a plant from seeds so selected that demonstrate improved germination under very high salt conditions and collecting seeds from the plant. 11. The method of claim 7, wherein the GPT transgene contains a GPT coding sequence that hybridizes to a GPT polynucleotide that encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15, SEQ ID NO: 17; SEQ ID NO: 19, SEQ ID NO: 21, under hybridization conditions that include hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37 ° C and at least one wash in 0.2 X SSC at a temperature of at least at least about 50 ° C for 20 minutes. 12. The method according to claim 7, where the transgenic plant is tobacco, tomato, pepper, beans, wigna, alfalfa, cantaloupe, pumpkin and saffron saffron. 13. The method of claim 7, wherein the transgenic plant has an increased leaf: GPT root ratio of activity compared to a plant of the same species that does not contain a GPT transgene or a GS transgene. 14. The method of claim 7, wherein the transgenic plant has an increased leaf: GS root ratio of activity compared to a plant of the same species that does not contain a GPT transgene or a GS transgene. 15. The method of claim 7, wherein the transgenic plant has an increased leaf: root ratio of 2-oxoglutomate compared to a plant of the same species that does not contain a GPT transgene or a GS transgene. 16. The transgenic plant according to claim 1, where the transgenic plant contains at least one additional growth characteristic compared to a wild-type analog or untransformed plant selected from the group consisting of earlier flowering, earlier budding, increased plant height, enhanced flowering increased budding, larger leaves, an increased yield of fruits or pods, and an increased yield of seeds. 17. The transgenic plant according to claim 1, where the transgenic plant has an increased ratio of leaf: root GPT activity compared to a plant of the same species that does not contain a GPT transgene or GS transgene. 18. The transgenic plant of claim 1, wherein the transgenic plant has an increased leaf: GS root ratio of activity compared to a plant of the same species that does not contain a GPT transgene or GS transgene. 19. The transgenic plant according to claim 1, wherein the transgenic plant has an increased leaf: root ratio of 2-oxoglutomat compared to a plant of the same species that does not contain a GPT transgene or a GS transgene. 20. The transgenic seed according to claim 6, where the plant grown from seeds exhibits increased tolerance to salt or salt conditions. 21. The transgenic seed of claim 6, wherein the GPT and GS transgenes are included in the transgenic seed genome. 22. The transgenic seed according to claim 6, where the transgenic seed is monocotyledonous. 23. The transgenic seed according to claim 6, where the transgenic seed is dicotyledonous. 24. A method for producing transgenic plants having increased production of 2-oxoglutaramate by transforming plant cells with transgenes that encode 2-oxoglutamate synthesis pathway enzymes, thereby increasing the biomass of transgenic plants compared to a wild-type analog or untransformed plant, comprising:
(a) introducing a transgene of glutamine-phenylpyruvate transaminase (GPT) into a first plurality of plants to produce a first plurality of transgenic plants;
(b) introducing a glutamine synthetase (GS) transgene into a second plurality of plants to produce a second plurality of transgenic plants;
(c) selecting a first plant from a first plurality of transgenic plants or their progeny, said plant containing a GPT transgene;
(d) selecting a second plant from a second plurality of transgenic plants or their progeny, said plant containing a GS transgene;
(e) crossing said first plant with said second plant to produce a double transgenic offspring containing said GPT transgene and said GS transgene,
wherein said double transgenic progeny produces more than 2-oxoglutamate relative to a plant of the same species that does not contain a GPT transgene or a GS transgene,
where the GPT transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, and
where the GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4 and SEQ ID NO: 7. 25. The method of claim 24, wherein the GPT transgene contains a GPT coding sequence that hybridizes to a GPT polynucleotide that encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15, SEQ ID NO: 17; SEQ ID NO: 19, SEQ ID NO: 21, under hybridization conditions, which include hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37 ° C and at least one wash in 0.2 X SSC at a temperature of at least at least about 50 ° C for 20 minutes. 26. The method according to p. 24, where the specified double-transgenic offspring contains at least one additional growth characteristic selected from the group consisting of increased biomass, earlier flowering, earlier budding, increased plant height, enhanced flowering, enhanced budding, more leaf size, increased yield of fruits or pods, and increased seed yield. 27. The method according to p. 24, additionally containing pollination of the offspring of the specified first set of transgenic plants to the stage of selection of the specified first plants from the offspring of the first set of transgenic plants. 28. The method according to p. 24, additionally containing pollination of the offspring of the specified second set of transgenic plants to the stage of selection of the specified second plants from the offspring of the second set of transgenic plants. 29. The method according to p. 24, where the first and second sets of plants are tobacco, tomato, pepper, beans, wigna, alfalfa, cantaloupe, pumpkin and saffron saffron. 30. The method according to p. 24, where the double-transgenic offspring has an increased ratio of leaf: GPT root activity compared to a plant of the same species that does not contain a GPT transgene or GS transgene. 31. The method of claim 24, wherein the double transgenic offspring has an increased leaf: GS root ratio of activity compared to a plant of the same species that does not contain a GPT transgene or GS transgene. 32. The method of claim 24, wherein the double transgenic offspring has an increased leaf: root ratio of 2-oxoglutomate compared to a plant of the same species that does not contain a GPT transgene or a GS transgene. 33. A method of producing transgenic plants having increased production of 2-oxoglutaramate by transforming plant cells with transgenes that encode 2-oxoglutamate synthesis pathway enzymes, thereby increasing the biomass of transgenic plants compared to a wild-type analog or untransformed plant, comprising:
(a) introducing (i) the transgene of glutamine-phenylpyruvate transaminase (GPT) and (ii) the transgene of glutamine synthetase (GS) in the first set of plant cells;
(b) the regeneration of many transgenic plants from plant cells; and
(c) selecting a transgenic plant having improved growth characteristics compared to a plant of the same species that does not contain a GPT transgene or GS transgene, where said transgenic plant produces more than 2-oxoglutamate relative to a plant of the same species that does not contain a GPT transgene or transgene GS
where the GPT transgene contains a GPT coding sequence that hybridizes to a GPT polynucleotide that encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15, SEQ ID NO: 17; SEQ ID NO: 19, SEQ ID NO: 21, under hybridization conditions, which include hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37 ° C and at least one wash in 0.2 X SSC at a temperature of at least at least about 50 ° C for 20 minutes,
The GS transgene encodes a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 4 and SEQ ID NO: 7. 34. The method according to p. 33, where improved growth characteristics are selected from the group consisting of increased biomass, earlier flowering, earlier budding, increased plant height, enhanced flowering, enhanced budding, larger leaves, increased yield of fruits or pods and increased seed crop. 35. The method according to p. 33, where the transgenic plant is tobacco, tomato, pepper, beans, wigna, alfalfa, cantaloupe, pumpkin and saffron saffron. 36. The method according to p. 33, where the transgenic plant has an increased ratio of leaf: root GPT activity compared to a plant of the same species that does not contain a GPT transgene or GS transgene. 37. The method of claim 33, wherein the transgenic plant has an increased leaf: GS root ratio of activity compared to a plant of the same species that does not contain a GPT transgene or a GS transgene. 38. The method of claim 33, wherein the transgenic plant has an increased leaf: root ratio of 2-oxoglutomat compared to a plant of the same species that does not contain a GPT transgene or a GS transgene. 39. The method of claim 33, further comprising collecting seeds from a transgenic plant and selecting a seed that exhibits improved germination under conditions of increased salt content. 40. The method according to p. 33, further comprising germinating the plant from a seed, showing improved germination under conditions of high salt content, and collecting its seeds.

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