ES2364988T3 - GRG23 EPSP IMPROVED SYNTHESES: COMPOSITIONS AND METHODS OF USE. - Google Patents

Abstract

A nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule containing the nucleotide sequence of SEQ ID NOs: 28, 30, 32, or 34; and, b) a nucleic acid molecule encoding a polypeptide containing the amino acid sequence of SEQ ID NOs: 29, 31, 33, or 35.

Description

Field of the Invention

This invention relates to molecular biology of plants, particularly to new EPSP synthase polypeptides that confer improved resistance or tolerance to glyphosate herbicide.

Background of the invention

N-phosphonomethylglycine, commonly referred to as glyphosate, in an important agronomic chemical. Glyphosate inhibits the enzyme that converts phosphoenolpyruvic acid (PEP) and 3-phosphoshichemical acid (S3P) into 5-enolpiruvil-3-phosphoshichemical acid. Inhibition of this enzyme (5-enolpiruvilshiquimato-3-phosphate synthase, referred to herein as "EPSP synthase", or "EPSPS") kills plant cells by closing the shiquimate pathway, thereby inhibiting the biosynthesis of aromatic amino acids.

Since glyphosate class herbicides inhibit the biosynthesis of aromatic amino acids, they not only kill plant cells, but are also toxic to bacterial cells. Glyphosate inhibits many bacterial EPSP synthases, and is therefore toxic to these bacteria. However, certain EPSP bacterial synthases have a high tolerance to glyphosate.

Plant cells resistant to glyphosate toxicity can be produced by transforming plant cells to express glyphosate-resistant bacterial EPSP synthases. Notably, the bacterial gene of the CP4 strain of Agrobacterium tumefaciens has been used to confer bactericidal resistance to plant cells after plant expression. A mutated EPSP synthase of strain CT7 of Salmonella typhimurium confers resistance to glyphosate in bacterial cells, and confers resistance to glyphosate on plant cells (US Pat. Nos. 4,535,060; 4,769,061; and 5,094,945).

US Patent No. 6,040,497 reports corn EPSP synthase mutant enzymes that have threonine substitutions for isoleucine at position 102 and proline for serine at position 106 (the "TIPS" mutation). Such alterations confer resistance to glyphosate on the corn enzyme. A mutated EPSP synthase of strain CT7 of Salmonella typhimurium confers resistance to glyphosate in bacterial cells, and is reported to confer resistance to glyphosate on plant cells (US Pat. Nos. 4,535,060; 4,769,061; and 5,094,945 ). He et al. ((2001) Biochim et Biophysica Acta 1568: 1-6) have developed EPSP synthases with greater glyphosate tolerance through mutagenesis and recombination between genes for EPSP synthase from E. coli and Salmonella typhimurium, and suggests that mutations in position 42 (T42M) and position 230 (Q230K) are probably responsible for the observed resistance. The following work (He et al. (2003) Biosci. Biotech. Biochem. 67: 1405-1409) shows that the T42M mutation (threonine by methionine) is sufficient to improve the tolerance of both the E. coli enzyme and the Salmonella typhimurium enzyme. Due to the many advantages that provide herbicide resistance to plants, herbicide resistance genes with enhanced glyphosate resistance activity are desirable.

An alternative method for mutagenesis is the "permutational mutagenesis" method described in US Patent Application No. 60 / 813,095, filed June 13, 2006.

Summary of the Invention

Compositions and methods for conferring resistance or tolerance to herbicides are provided. The compositions include EPSP synthase enzymes that are resistant to glyphosate herbicide, and nucleic acid molecules encoding such enzymes, vectors containing those nucleic acid molecules, and host cells containing vectors. The compositions include nucleic acid molecules encoding herbicide resistance polypeptides, including those encoding polypeptides comprising SEQ ID NOs: 29, 31, 33, or 35, as well as the polynucleotide sequences of SEQ ID NOs: 28 , 30, 32, or 34.

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Therefore, the present invention provides a nucleic acid molecule selected from the group consisting of from:

a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs: 28, 30, 32, or 34; Y, b) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of the SEQ ID NOs: 29, 31, 33, or 35.

The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The compositions too they include bacteria, plants, plant cells, tissues, and transformed seeds that are resistant to glyphosate by means of introducing the compositions of the invention into the genome of organisms. Where the organism it is a plant, the introduction of the sequence allows glyphosate containing herbicides to be applied to plants to selectively kill weeds sensitive to glyphosate or other non-transformed plants, but not the transformed organism. The sequences can be additionally used as markers for selection of plant cells that grow under glyphosate conditions.

Methods for identifying an EPSP synthase enzyme with resistance activity are also provided. glyphosate The methods comprise the identification of additional sequences of EPSP synthase that are resistant to glyphosate based on the presence of the domain of the invention.

The present invention also provides:

- a vector containing the nucleic acid molecule of the invention, preferably also containing a nucleic acid molecule encoding a heterologous polypeptide;

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a host cell containing the nucleic acid molecule of the invention, preferably a bacterial host cell or a plant cell;

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a transgenic plant containing the host cell of the plant of the invention, preferably wherein said plant is selected from the group consisting of corn, sorghum, wheat, sunflower, tomato, cruciferous, peppers, potato, cotton, rice, soybeans, beets sugar, sugarcane, tobacco, barley, and oilseed rape; Y

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a transgenic seed containing the nucleic acid molecule of the invention.

Additionally, the invention provides a recombinant polypeptide selected from the group consisting of:

a) a polypeptide containing the amino acid sequence of SEQ ID NOs: 29, 31, 33, or 35; Y, b) a polypeptide encoded by the nucleotide sequence of SEQ ID NOs: 28, 30, 32, or 34, preferably which also contains a heterologous amino acid sequence.

The invention also provides a method for conferring resistance to a herbicide in a plant, said method of transforming said plant with a DNA construct, said construct containing a promoter that directs expression in a plant cell operatively linked to a sequence of nucleotides selected from the group consisting of SEQ ID NOs: 28, 30, 32, or 34, and regenerate a plant transformed, preferably where said herbicide is glyphosate.

The invention also provides a plant that has a stable construction in its genome. DNA that contains a nucleotide sequence that encodes a protein that has resistance activity to herbicides, wherein said nucleotide sequence is selected from the group consisting of:

a) a nucleic acid molecule containing the nucleotide sequence of SEQ ID NOs: 28, 30, 32, or 34; Y, b) a nucleic acid molecule that encodes a polypeptide that contains the amino acid sequence of SEQ ID NOs: 29, 31, 33, or 35; wherein said nucleotide sequence is operably linked to a promoter that directs the expression of a coding sequence in a cell of a plant.

Additionally, the invention provides a method for increasing vigor or productivity in a plant that understands:

a) provide a plant containing the nucleotide sequence set forth in SEQ ID NOs: 28, 30, 32, or 34; b) contacting said plant with an effective glyphosate concentration; Y, c) cultivate said plant under conditions where the temperature is higher than the ambient temperature at least for two consecutive hours per day for at least four days after contact with said glyphosate, where these days after the contact are within the vegetative period of the plant, where the vigor or productivity of said plant are greater than the vigor or productivity of a plant that expresses a glyphosate tolerance EPSP synthase that does not have an optimum temperature higher than the ambient temperature, preferably where the temperature in step (c) is approximately 32 ° C to approximately 60 ° C.

The invention also provides a method for conferring glyphosate resistance in a plant comprising:

a) provide a plant containing the nucleotide sequence set forth in SEQ ID NOs: 28, 30, 32, or 34; b) contacting said plant with an effective glyphosate concentration; Y, c) cultivate said plant under conditions where the temperature is higher than the ambient temperature at least for two consecutive hours per day for at least four days after contact with said glyphosate, where these days after the contact are within the vegetative period of the plant.

Description of the figures

Figure 1 demonstrates the enzymatic activity of GRG23 (acel) ("M5"; SEQ ID NO: 10) compared to GRG23 of wild type ("WTGRG23"; SEQ ID NO: 2) at 37 ° C for 0 to 25 hours. Figure 2 shows the resistance of GRG23 and different variants to glyphosate, expressed as the constant of inhibition, or K1. Figure 3 shows the half-life of GRG23 and variants of GRG23 at 37 ° C.

Detailed description of the invention

The present inventions will be described hereinafter more fully with reference to the drawings companions, in which some but not all modalities of the inventions are presented. Actually, These inventions can be incorporated in many different ways and should not be construed as limited to the modalities set forth here; rather, these modalities are presented in such a way that this disclosure meet applicable legal requirements. Throughout the description, similar numbers refer to similar elements.

Many modifications and other modalities of the inventions set forth herein will come to the mind of the person. trained in the art to which these inventions belong having the benefit of the teachings presented in the previous descriptions and in the associated drawings. Therefore, it is understood that inventions are not limited to the specific modalities described and what modifications and other modalities are intended to be included within the scope of the appended claims. Although specific terms are used here, they are used only in a generic and descriptive sense and not for purposes of limitation.

The present invention is directed to compositions and methods for regulating herbicide resistance in organisms, particularly in plants or in plant cells. The methods involve the transformation of organisms with nucleotide sequences encoding a glyphosate resistance gene of the invention. The nucleotide sequences of the invention are useful for the preparation of plants that show greater tolerance to glyphosate herbicide. Therefore, by "glyphosate resistance" or "glyphosate tolerance" gene of the invention the nucleotide sequence set forth in SEQ ID NOs: 28, 30, 32, or 34, and fragments and variants of the same that encode a polypeptide of resistance or tolerance to glyphosate. Similarly, a polypeptide of "glyphosate resistance" or "glyphosate tolerance" of the invention is a polypeptide having the sequence of

amino acids set forth in SEQ ID NOs: 29, 31, 33, or 35, and fragments and variants thereof, which confer resistance or tolerance to glyphosate to a host cell.

A. Isolated polynucleotides, and variants and fragments thereof

In some embodiments, the present invention includes isolated, recombinant, or purified polynucleotides. An "isolated" or "purified" polynucleotide or polypeptide, or a biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or of other chemical compounds when chemically synthesized. By "biologically active" is meant to possess the desired biological activity of the native polypeptide, that is, to retain the herbicide resistance activity. An "isolated" polynucleotide can be free of sequences (for example, of sequences encoding protein) that naturally flank the nucleic acid (ie, sequences located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the polynucleotide is derived. For the purposes of the invention, "isolated" when used to refer to polynucleotides excludes isolated chromosomes. For example, in different embodiments, the isolated polynucleotide encoding glyphosate resistance may contain approximately less than 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of the sequence of nucleotides that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived.

The polynucleotides of the invention include those encoding glyphosate resistance polypeptides containing SEQ ID NOs: 29, 31, 33, or 35, as well as the polynucleotide sequences of SEQ ID NOs: 28, 30, 32, or 34 .

By "glyphosate" is meant any herbicidal form of N-phosphonomethylglycine (including any salt thereof) and other forms that result in the production of glyphosate anion in the plant. A "herbicide resistance protein"

or a protein that results from the expression of a "nucleic acid molecule that encodes herbicide resistance" includes proteins that give a cell the ability to tolerate a higher concentration of a herbicide than cells that do not express the protein, or to tolerate a certain concentration of a herbicide for a longer period of time than cells that do not express the protein. A "glyphosate resistance protein" includes a protein that gives a cell the ability to tolerate a higher concentration of glyphosate than cells that do not express the protein, or to tolerate a certain concentration of glyphosate for a longer period of time. long than cells that do not express protein. By "tolerate" or "tolerance" is meant either to survive, or to carry out essential cellular functions such as protein synthesis and respiration in a way that is not readily discernible from untreated cells.

The present invention further contemplates variants and fragments of the polynucleotides described herein. A "fragment" of a polynucleotide can encode a biologically active portion of a polypeptide, or it can be a fragment that can be used as a hybridization probe or an initiator for PCR using methods disclosed herein elsewhere. Polynucleotides that are fragments of a polynucleotide include approximately minus 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 , 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950 contiguous nucleotides, or even the number of nucleotides present in a full length polynucleotide disclosed herein depending on the intended use (for example, an EPSP synthase polynucleotide comprising SEQ ID NO: 1). "Adjacent" nucleotides are understood as nucleotide residues that are immediately adjacent to each other.

Fragments of the polynucleotides of the present invention will generally encode polypeptide fragments that retain the biological activity of the full-length glyphosate resistance protein; that is, herbicide resistance activity. "Retains herbicide resistance activity" means that the fragment has approximately at least 30%, approximately at least 50%, approximately at least 70%, approximately at least 80%, 85%, 90%, 95%, 100% , 110%, 125%, 150%, 175%, 200%, 250%, approximately at least 300% or more of the herbicide resistance activity of the full-length glyphosate resistance protein disclosed herein as SEQ ID NOs : 2, 3, or 5. Methods for measuring herbicide resistance activity are well known in the art. See, for example, United States Patents Nos. 4,535,060, and 5,188,642.

A fragment of a polynucleotide encoding a biologically active portion of a polypeptide of the invention will encode at least 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400 contiguous amino acids , or up to the total number of amino acids present in a full length polypeptide of the invention.

The invention also encompasses variants of the polynucleotides. The "variants" of polynucleotides include those sequences that encode the polypeptides disclosed herein but differ conservatively due to degeneracy of the genetic code, as well as those that are sufficiently identical.

The term "sufficiently identical" encompasses a polypeptide or polynucleotide sequence having approximately at least 60% or 65% sequence identity, approximately 70% or 75% sequence identity, approximately 80% or 85% sequence identity, approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using one of the alignment programs using standard parameters . Someone skilled in the art will realize that these values can be appropriately adjusted to determine the corresponding identity of the polypeptides encoded by two polynucleotides taking into account codon degeneration, amino acid similarity, reading frame positioning, and the like. .

Bacterial genes very often have multiple methionine initiation codons in the vicinity of the beginning of the open reading frame. Often, initiation of translation into one or more of these start codons will lead to the generation of a functional protein. These start codons may include ATG codons. However, bacteria such as Bacillus sp. they also recognize the GTG codon as a start codon, and the proteins that initiate translation in the GTG codons contain a methionine in the first amino acid. In addition, it is often not determined a priori which of these codons are used naturally in the bacteria. Therefore, it is understood that the use of one of the alternative methionine codons can lead to the generation of variants that confer resistance to herbicides. These herbicide resistance proteins are included in the present invention and can be used in the methods of the present invention.

Allelic variants of natural origin can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as indicated below. The polynucleotide variants also include polynucleotides of synthetic origin that have been generated, for example, through the use of site-directed mutagenesis strategies or other mutagenesis strategies but still encode the polypeptide that has the desired biological activity.

The skilled person will also realize that changes can be introduced by means of an additional mutation of the polypeptides of the invention thus leading to additional changes in the amino acid sequence of the encoded polypeptides, without altering the biological activity of the polypeptides. Therefore, isolated polynucleotide variants can be created by introducing one or more additional nucleotide substitutions, additions or deletions into the corresponding polynucleotide encoding the EPSP synthase domain disclosed herein, such that a or more amino acid substitutions, additions or deletions in the encoded polypeptide. Additional mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis, or gene exchange techniques. Such variants of the polynucleotides are also encompassed by the present invention.

Polynucleotide variants can be made by introducing random mutations throughout all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be selected for the ability to confer activity of herbicide resistance to identify mutants that retain activity.

Gene rearrangement or sexual PCR procedures can be used (eg, Smith (1994) Nature

370: 324-325; United States Patents Nos. 5,837,458; 5,830,721; 5,811,238; and 5,733,731), to further modify or improve polynucleotides and polypeptides having the EPSP synthase domain of the present invention (eg, polypeptides that confer glyphosate resistance). The rearrangement of genes involves random fragmentation of different mutant DNAs followed by their new PCR assembly in full-length molecules. Examples of different gene rearrangement procedures include, but are not limited to assembly followed by DNase treatment, the stepwise extension process (STEP), and in vitro random priming recombination. In the DNase mediated method, isolated segments of DNA are cleaved from a reservoir of positive mutants in random fragments with DNase I and subjected to multiple rounds of PCR without initiator addition. The lengths of the random fragments approximate those of the non-cleaved segment as the PCR cycles progress, resulting in mutations in different clones mixing and accumulating in some of the resulting sequences. Multiple selection and ordering cycles have led to the functional improvement of different enzymes (Stemmer (1994) Nature 370: 389 391; Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751; Crameri et al. (1996 ) Nat. Biotechnol. 14: 315 319; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4504-4509; and Crameri et al. (1997) Nat. Biotechnol. 15: 436

- 438). Such procedures could be carried out, for example, on polynucleotides encoding polypeptides that have the domain of the sequence of the present invention to generate polypeptides that confer glyphosate resistance.

Using methods such as PCR, hybridization, and the like, the corresponding herbicide resistance sequences can be identified by searching for domains of EPSP synthase of the present invention. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Innis et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY).

B. Isolated proteins and variants and fragments thereof could

Polypeptides for herbicide resistance are also encompassed within the present invention. A herbicide resistance polypeptide that is substantially free of cellular material includes in polypeptide preparations that have approximately less than 30%, 20%, 10%, or 5% (dry weight) of a polypeptide that is not for resistance to herbicides (also referred to herein as a "contaminant"). In the present invention, "herbicide resistance protein" includes an EPSP synthase polypeptide having the sequence domain of the invention. Fragments, biologically active portions, and variants thereof are also provided, and can be used to carry out the methods of the present invention.

"Fragments" or "biologically active portions" include polypeptide fragments that contain a portion of an amino acid sequence that encodes a protein for herbicide resistance and that retains herbicide resistance activity. A biologically active portion of a protein for herbicide resistance may be a polypeptide that is, for example, 10, 25, 50, 100 or more amino acids in length. Such biologically active portions can be prepared by means of recombinant techniques and evaluated by herbicide resistance activity.

By "variants" is meant proteins or polypeptides having an amino acid sequence that is approximately at least 60%, 65%, approximately 70%, 75%, approximately 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% identical to an EPSP synthase polypeptide having the EPSP synthase domain of the present invention. Someone skilled in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of polypeptides encoded by two polynucleotides taking into account codon degeneration, amino acid similarity, reading frame positioning, and the like.

For example, conservative amino acid substitutions can be made in one or more non-essential amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild type sequence of a polypeptide without altering the biological activity, while an "essential" amino acid residue is required for the biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, Threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and side chains aromatic (for example, tyrosine, phenylalanine, tryptophan, histidine). Amino acid substitutions can be made in unconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential for polypeptide activity. However, someone skilled in the art would understand that functional variants may have minor alterations conserved or not conserved in conserved residues.

Also included are antibodies to the polypeptides of the present invention, or to variants or fragments thereof. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; U.S. Patent No. 4,196,265 ).

C. Polynucleotide constructs

The polynucleotides encoding the EPSP synthase polypeptides of the present invention can be modified to obtain or improve expression in plant cells. The polynucleotides encoding the polypeptides identified herein can be supplied in expression cassettes for expression in the plant of interest. A "plant expression cassette" includes a DNA construct, which includes a recombinant DNA construct, which is capable of resulting in the expression of a polynucleotide in a plant cell. The cassette may include in the 5'-3 'transcription direction, a transcriptional initiation region (i.e., promoter, particularly a heterologous promoter) operably linked to one or more polynucleotides of interest, and / or a transcriptional termination region and of translation (that is, a termination region) functional in plants. The cassette may additionally contain at least one additional polynucleotide to be introduced into the body, such as a selectable marker gene. Alternatively, the additional polynucleotide (s) can be supplied on multiple expression cassettes. Such an expression cassette has a plurality of restriction sites for insertion of the polynucleotide (s) to be under the transcriptional regulation of the regulatory regions.

"Heterologists" generally refers to the polynucleotide or polypeptide that is not endogenous to the cell or that is not endogenous to the location in the native genome in which it is present, and has been added to the cell by infection, transfection, microinjection, electroporation , microprojection, or the like. By "operably linked" is meant a functional link between two polynucleotides. For example, when a promoter is operably linked to a DNA sequence, the promoter sequence initiates and mediates the transcription of the DNA sequence. It is recognized that operably linked polynucleotides may or may not be contiguous and, where they are used as a reference the binding of two regions encoding a polypeptide, the polypeptides are expressed in the same reading frame.

The promoter can be any polynucleotide sequence that shows transcriptional activity in plant cells, parts of the plant, or selected plants. The promoter can be native or analogous, or external or heterologous, to the host plant and / or to the DNA sequence of the invention. Where the promoter is "native" or "analogous" to the host plant, it is understood that the promoter is within the native plant into which the promoter is introduced. Where the promoter is "external" or "heterologous" to the DNA sequence of the invention, it is understood that the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention. The promoter can be inducible or constitutive. It can be of natural origin, it can be composed of portions of different promoters of natural origin, or it can be partially or totally synthetic. Guidance for promoter design is provided by promoter structure studies, such as those by Harley and Reynolds (1987) Nucleic Acids Res. 15: 2343-2361. Also, promoter location can be optimized relative to Start of transcription. See, for example, Roberts et al. (1979) Proc. Natl Acad. Sci. USA,

76: 760-764. Many promoters suitable for use in plants are well known in the art.

For example, constitutive promoters suitable for use in plants include: plant virus promoters, such as the peanut chlorotic vein caulimovirus (PClSV) promoter (U.S. Patent No. 5,850,019); the 35S promoter of cauliflower mosaic virus (CaMV) (Odell et al. (1985) Nature 313: 810-812); gene promoters for Chlorella virus methyltransferase (U.S. Patent No. 5,563,328) and the full length transcript promoter of scrofular mosaic virus (FMV) (U.S. Patent No. 5,378,619) ; gene promoters such as rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3: 2723-2730); H3 histone from corn (Lepetit et al. (1992) Mol. Gen. Genet. 231: 276-285 and Atanassova et al. (1992) Plant J. 2 (3): 291-300); ALS3 of Brassica napus (PCT Application WO 97/41228); and promoters of different Agrobacterium gens (see US Patents Nos. 4,771,002; 5,102,796; 5,182,200; and 5,428,147).

Inducible promoters suitable for use in plants include: the promoter of the ACE 1 system that responds to copper (Mett et al. (1993) PNAS 90: 4567-4571); to the In2 gene promoter of maize that responds to the protectors of the benzene sulfonamide herbicide (Hershey et al. (1991) Mol. Gen. Genetics 227: 229-237 and Gatz et al. (1994) Mol. Gen. Genetics 243: 32 - 38); and the Tet repressor promoter of Tn10 (Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237). Another inducible promoter for use in plants is one that responds to an induction agent for which plants normally do not respond. An example of such an inducible promoter is the inducible promoter of a gene for a steroid hormone, whose transcriptional activity is induced by a glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421 ) or the recent application of a chimeric transcription activator, XVE, for use in an inducible expression system of a plant based on the estrogen receptor activated by estradiol (Zuo et al. (2000) Plant J., 24: 265 - 273). Other inducible promoters for use in plants are described in EP 332104, PCT WO 93/21334 and PCT WO 97/06269. Promoters composed of portions of other promoters and promoters totally or partially synthetic may also be used. See, for example, Ni et al. (1995) Plant J. 7: 661-676 and PCT WO 95/14098 describing such promoters for use in plants.

The promoter may include, or be modified to include, one or more reinforcing elements. In some embodiments, the promoter may include a plurality of reinforcing elements. Promoters containing precursor elements provide higher levels of transcription compared to promoters that do not include them. Reinforcing elements suitable for use in plants include the PClSV reinforcing element (United States Patent No. 5,850,019), the CaMV 35S reinforcing element (United States Patents Nos. 5,106,739 and 5,164,316) and to the FMV reinforcing element (Maiti et al. (1997) Transgenic Res. 6: 143-156). See also PCT WO 96/23898.

Often, such constructions may contain 5 ’and 3’ untranslated regions. Such constructs may contain a "signal sequence" or "leader sequence" to facilitate transport at time with translation or after translation of the peptide of interest to certain intracellular structures such as chloroplast (or other plastid), to the endoplasmic reticulum, or to the Golgi apparatus, or to be secreted. For example, the construct can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum. By "signal sequence" is meant a sequence that is known or suspected to result in the transport of the peptide at the time of translation or after translation through the cell membrane. In eukaryotes, this typically involves secretion in the Golgi apparatus, with some resulting glycosylation. By "leader sequence" is meant any sequence that, when translated, results in a sufficient amino acid sequence to activate transport time with the translation of the peptide chain into a subcellular organelle. Therefore, this includes leading sequences that direct transport and / or glycosylation through passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. It may also be preferable to genetically modify the plant expression cassette to contain an intron, such that processing of the intron mRNA for expression is required.

By "untranslated region 3" means a polynucleotide located sequence below an encoding sequence. The polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting the addition of polyadenyl acid tracts to the 3 ′ end of the mRNA precursor are 3 ’untranslated regions. The term "untranslated region 5" means a polynucleotide located upstream of a coding sequence.

Other untranslated elements sequence up and sequence down include reinforcers. Enhancers are polynucleotides that act to increase the expression of a promoter region. Enhancers are well known in the art and include, but are not limited to, the SV40 reinforcing region and the 35S reinforcing element.

The termination region may be native to the transcription initiation region, may be native to the sequence of the present invention, or may be derived from another source. Suitable termination regions are available from the Ti plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.

In one aspect of the invention, synthetic DNA sequences are designed for a given polypeptide, such as the polypeptides of the invention. The expression of the open reading frame of the synthetic DNA sequence in a cell results in the production of the polypeptide of the invention. Synthetic DNA sequences can be useful to simply remove unwanted restriction endonuclease sites, to facilitate DNA cloning strategies, to alter or remove any potential codon bias, to alter or modify GC content, to remove or alter alternating reading frames, and / or to alter or remove intron / exon splice recognition sites, polyadenylation sites, Shine-Delgarno sequences, unwanted promoter elements and the like that may be present in a native DNA sequence. It is also possible that synthetic DNA sequences can be used to introduce other improvements to a DNA sequence, such as the introduction of an intron sequence, the creation of a DNA sequence that is expressed as a protein fusion with target sequences of the Organelle, such as chloroplast transit peptides, apoplast / vacuolar target peptides, or peptide sequences that result in retention of the resulting peptide in the endoplasmic reticulum. Synthetic genes can also be synthesized using preferred codons of the host cell for enhanced expression, or they can be synthesized using codons with a codon usage frequency preferred by the host. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11; United States Patents Nos. 6,320,100; 6,075,185; 5,380,831; and 5,436,391, Published American Applications Nos. 20040005600 and 20010003849, and Murray et al. (1989) Nucleic Acids Res. 17: 477-498.

In one embodiment, the polynucleotides of interest are directed to the chloroplast for expression. In this form, where the polynucleotide of interest is not directly inserted into the chloroplast, the expression cassette will additionally contain a polynucleotide encoding a transit peptide to direct the nucleotide of interest to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481.

The polynucleotides of interest that are directed to the chloroplast can be optimized for expression in the chloroplast to account for the differences in the use of the codon between the core of the plant and this organelle. In this way, the polynucleotides of interest can be synthesized using preferred chloroplast codons. See, for example, U.S. Patent No. 5,380,831.

This plant expression cassette can be inserted into a plant transformation vector. By "transformation vector" is meant a DNA molecule that allows the transformation of a cell. Such a molecule can consist of one or more expression cassettes, and can be organized into more than one vector DNA molecule. For example, binary vectors are plant transformation vectors that use two non-contiguous DNA vectors to encode all the essential functions that act in cis and trans form for plant cell transformation (Hellens and Mullineaux (2000) Trends in Plant Science 5: 446-451). "Vector" refers to a polynucleotide construct designed for transfer between different host cells. "Expression vector" refers to a vector that has the ability to incorporate, integrate and express heterologous DNA sequences or fragments in a foreign cell.

The plant transformation vector comprises one or more DNA vectors to achieve plant transformation. For example, it is a common practice in the art to use plant transformation vectors that include more than one contiguous segment of DNA. These vectors are often known in the art as binary vectors. Binary vectors as well as vectors with auxiliary plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of the DNA elements necessary to achieve efficient transformation is quite large, and it is convenient to separate functions on separate molecules. of DNA. Binary vectors typically contain a plasmid vector containing cis-acting sequences required for the transfer of T-DNA (such as the left edge and the right edge), a selectable marker that is engineered to be expressed in a gene. a plant cell, and a "polynucleotide of interest" (a genetically engineered polynucleotide to be expressed in a cell of a plant for which the generation of transgenic plants is desired). The sequences required for bacterial replication are also present on this plasmid vector. The sequences that act in cis form are arranged in a way that allows an efficient transfer within the cells of a plant and their expression in them. For example, the selectable marker sequence and the sequence of interest are located between the left and right edges. Often a second plasmid vector contains the trans-acting factors that mediate the transfer of Agrobacterium T-DNA to plant cells. This plasmid often contains virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and DNA transfer by cleavage in the edge sequences and vir-mediated DNA transfer, as understood in art (Hellens and Mullineaux (2000) Trends in Plant Science, 5: 446-451). Different types of Agrobacterium strains (for example, LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for the introduction of polynucleotides into plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.

D. Plant transformation

The methods of the invention involve the introduction of a nucleotide construct into a plant. By "introduction" is meant to present the nucleotide construction to the plant in such a way that the construction gains access to the interior of a plant cell. The methods of the invention do not require that a particular method be used to introduce a nucleotide construct to a plant, only that the nucleotide construct gain access to the interior of at least one plant cell. Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus mediated methods.

In general, plant transformation methods involve the transfer of heterologous DNA into target plant cells (e.g. mature or immature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by the application of a level of the appropriate maximum selection threshold (depending on the selectable marker gene and in this case "glyphosate") to coat the transformed plant cells of a group of a non-transformed mass of cells. Explants are typically transferred to a fresh supply of the same medium and routinely grown. Subsequently, the transformed cells differentiate into outbreaks after placing them on regeneration medium supplemented with a maximum threshold level of selection agent (eg "glyphosate"). The shoots are then transferred to a selective rooting medium to recover the rooted shoots or seedlings. Transgenic seedlings then grow to mature plants and produce fertile seeds (for example, Hiei et al. (1994) The Plant Journal 6: 271-282; Ishida et al. (1996) Nature Biotechnology 14: 745-750). Explants are typically transferred to a fresh supply of the same medium and routinely grown. A general description of the techniques and methods for generating transgenic plants is found in Ayres and Park (1994) Critical Reviews in Plant Science 13: 219-239 and Bommineni and Jauhar (1997) Maydica 42: 107-120. Since the transformed material It contains many cells, both transformed and non-transformed cells are present in any piece of callus or tissue or group of subjected target cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove transformed cells is a limitation to quickly recover the cells of transformed plants and the successful generation of transgenic plants. Molecular and biochemical methods can be used to confirm the presence of the integrated heterologous gene of interest in the genome of transgenic plants.

The generation of transgenic plants can be formed by one of different methods, including, but not limited to, the introduction of heterologous DNA through Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with DNA foreign heterologous adhered to particles, and other different methods not directly mediated by particles (for example Hiei et al. (1994) The Plant Journal

6: 271-282; Ishida et al. (1996) Nature Biotechnology 14: 745-750; Ayres and Park (1994) Critical Reviews in Plant Science 13: 219-239; Bommineni and Jauhar (1997) Maydica 42: 107-120) to transfer DNA.

The methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl Acad. Sci. USA 87: 8526-8530; Svab and Maliga (1993) Proc. Natl Acad. Sci. USA 90: 913-917; Svab and Maliga (1993) EMBO J. 12: 601-606. The method is based on the delivery by means of a DNA particle gun containing a selectable marker and directing the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be achieved by transactivating a transgene transmitted by a silent plastid by preferred expression in the tissue of an RNA polymerase directed to the plastid and encoded in the nucleus. Such a system has been reported in McBride et al. (1994) Proc. Natl Acad. Sci. USA

91: 7301-7305.

Cells that have been transformed can be grown to plants according to conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants can then be grown, and either pollinated with the same transformed strain or with different strains, and identified the resulting hybrid that has constitutive expression of the desired phenotypic characteristic. Two or more generations can be cultivated to ensure that the expression of the desired phenotypic characteristic is maintained in a stable manner, and inherited and then harvested the seeds to ensure that the expression of the desired phenotypic characteristic has been achieved. In this form, the present invention provides transformed seeds (also called "transgenic seeds") having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into its genome.

E. Evaluation of plant transformation

After the introduction of the heterologous foreign DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed by means of different methods such as the analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

PCR analysis is a quick method to select cells, tissue or buds transformed by the presence of the gene incorporated in the early stage before transplanting them in the soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)). PCR is carried out using oligonucleotide primers specific for the gene of interest or with the support of the Agrobacterium vector, etc.

Plant transformation can be confirmed by a Southern blot analysis of genomic DNA (Sambrook and Russell (2001), see above). In general, DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated on an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or the "transfer" can then be tested, for example, with a target DNA fragment radiolabelled with 32P to confirm the integration of the gene introduced into the plant genome according to standard techniques (Sambrook and Russell, 2001, see above).

In Northern analysis, RNA is isolated from specific tissues of the transformant, fractionated on an agarose / formaldehyde gel, and transferred onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell (2001), see above). The expression of the RNA encoded by grg sequences of the invention is then analyzed by hybridization of the filter with a radioactive probe derived from a GDC by means of methods known in the art (Sambrook and Russell (2001), see above).

Western blotting and biochemical assays and the like can be carried out on transgenic plants to determine the presence of protein encoded by the herbicide resistance gene by standard procedures (Sambrook and Russell (2001), see above) using antibodies which bind to one or more epitopes present on the herbicide resistance protein.

In one aspect of the invention, the grg genes described herein are useful as markers for assessing the transformation of bacterial or plant cells.

F. Plants and parts of plants

"Plant" means whole plants, plant organs (for example, leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and their progeny. Plant cells can be differentiated or undifferentiated (for example, callus, cells in suspension culture, protoplasts, leaf cells, root cells, phloem cells, pollen). The present invention can be used for the introduction of polynucleotides in any species of plant, including, but not limited to, monocot and dicot. Examples of plants of interest include, but are not limited to, corn, sorghum, wheat, sunflower, tomato, crucifers, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, and seed rape. oilseeds, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almonds, oats, vegetables, ornamental plants, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, green beans, beans, peas, and members of the Curcumis genus such as cucumber, cantaloupe and melon. Ornamental plants include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnations, poinsettia, and chrysanthemum. Also of interest are crop plants, including, for example, corn, sorghum, wheat, sunflower, tomato, cruciferous, peppers, potato, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, oilseed rape , etc.

This invention is suitable for any member of the family of monocot plants including, but not limited to, corn, rice, barley, oats, wheat, sorghum, rye, sugarcane, pineapple, stamens, onion, banana, coconut, and dates

G. Methods to increase plant productivity

Methods are provided to increase plant productivity. The methods comprise the introduction into a plant or a cell of a plant of a polynucleotide containing a grg sequence disclosed herein. As defined here, the "productivity" of the plant refers to the quality and / or quantity of biomass produced by the plant. "Biomass" means any measurable plant product. An increase in biomass production is any improvement in the productivity of the measured vegetable product. The increase in plant productivity has different commercial applications. For example, the increase in plant leaf biomass can increase the productivity of leaf vegetables for human or animal consumption. Additionally, the increase in leaf biomass can be used to increase the production of industrial or pharmaceutical products derived from plants. An increase in productivity may include any statistically significant increase including, but not limited to, at least a 1% increase, at least a 3% increase, at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 50% increase, at least a 70% increase, at least a 100% increase or a higher increase.

In specific methods, the plant is treated with an effective concentration of a herbicide, where the application of the herbicide results in increased plant productivity. "Effective concentration" means the concentration that allows the highest productivity in the plant. Such effective concentrations for herbicides of interest are generally known in the art. The herbicide can be applied either before or after the plants emerge according to usual techniques for applying herbicides to fields containing crops that have become resistant to herbicides by means of heterologous expression of a grg gene from invention.

Methods of conferring herbicide resistance in a plant or part of a plant are also provided. In such methods, a grg polynucleotide disclosed herein is introduced into the plant, where polynucleotide expression results in tolerance or resistance to glyphosate. Plants produced through this method can be treated with an effective concentration of a herbicide and show a greater tolerance to the herbicide. An "effective concentration" of a herbicide in this application is an amount sufficient to slow or stop the growth of plants or parts of plants that are not naturally resistant or that became resistant to the herbicide.

H. Methods to control weeds in a field

Methods for selectively controlling weeds in a field that contains a plant are also provided. In one embodiment, the seeds of a plant or plants are resistant to glyphosate as a result of the insertion of a grg polynucleotide disclosed herein in the seed of the plant or in the plant. In specific methods, the plant is treated with an effective concentration of a herbicide, where the application of the herbicide results in a selective control of weeds or other non-transformed plants. "Effective concentration" means the concentration that controls the growth or spread of weeds or other non-transformed plants without significantly affecting the seed of the plant or the glyphosate resistant plant. Such effective concentrations for herbicides of interest are generally known in the art. The herbicide can be applied either before or after the plant emerges according to usual techniques for applying herbicides to fields containing plants or plant seeds that have become resistant to the herbicide.

1. Temperature spectrum

Different studies of glyphosate metabolism in plants have been carried out, and revealed that glyphosate is not metabolized by plants or is metabolized very slowly. Glyphosate quickly penetrates the cuticle, and travels throughout plants for a considerable period of time (reviewed in Kearney and Kaufman, Eds (1988) Herbicides; Chemistry, Degradation & Mode of Action Marcel Dekker, Inc., New York , 3: 1-70 and Grossbard and Atkinson, Eds. (1985) The Herbicide Glyphosate Butterworths, London, pages 25-34). Therefore, glyphosate tolerance is likely to be necessary for a prolonged period of time followed by exposure to glyphosate in agronomically important plants. Where temperatures frequently exceed 30 ° C during the vegetative period, it would be convenient to use a tolerance EPSP synthase that maintains its activity at elevated temperatures.

In one embodiment of the present invention, EPSP synthase exhibits thermal stability at a temperature that is higher or lower than the ambient temperature. By "thermal stability" is meant that the enzyme is active at a temperature higher or lower than room temperature for a longer period of time than an EPSP synthase that is not thermally stable at that temperature. For example, thermally stable EPSP synthase has enzymatic activity for approximately more than 1 hour, approximately for more than 2 hours, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11 , about 12, about 13, about 14, about 15, about 20, about 25 hours, or more, at a temperature that is higher or lower than room temperature. For the purposes of the present invention, the "ambient" temperature is approximately 30 ° C. In some embodiments, a temperature above ambient is a temperature of approximately 32 ° C, approximately 34 ° C, approximately 37 ° C, approximately 40 ° C, approximately 45 ° C, approximately 50 ° C, approximately 55 ° C, about 60 ° C, about 65 ° C, or higher. A temperature below ambient is a temperature of approximately or below 28 ° C, approximately below 27 ° C, approximately 26 ° C, approximately 25 ° C, approximately 23 ° C, approximately 20 ° C, approximately 18 ° C, about 15 ° C, about 10 ° C, about or below 5 ° C, or about 0 ° C. The methods to analyze the activity of EPSP synthase are discussed in more detail here elsewhere. For the purposes of the present invention, a thermally active EPSP synthase is considered when it operates approximately 90% to 100%, approximately 80% to approximately 90%, approximately 70% to approximately 80%, approximately 60% up to about 70% or about 50% to about 60% of the maximum activity level observed at the optimum temperature for that enzyme.

Therefore, methods and compositions for increasing glyphosate tolerance at temperatures above room temperature are provided herein. In one embodiment, the methods comprise the introduction into a plant of a nucleotide sequence encoding the EPSP synthase enzyme for glyphosate tolerance set forth in SEQ ID NOs: 8, 10, 14, 28, 30, 32, or 34, and plant cultivation at a temperature above room temperature. In specific embodiments, the culture temperature is higher than room temperature for an average of at least about 2 hours per day, at least about 3 hours per day, at least about 4 hours per day, at least about 5, about 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 14, approximately 16, approximately 18, approximately 20, at least approximately 22 hours per day, or up to approximately 24 hours per day during the vegetative period of plant.

In another embodiment, the method comprises the introduction into a plant of a nucleotide sequence that encodes the EPSP synthase enzyme for glyphosate tolerance set forth in SEQ ID NOs: 8, 10, 14, 28, 30, 32, or 34, setting contact the plant with an effective glyphosate herbicide concentration, and cultivating the plant at a temperature that exceeds room temperature for at least 1 hour, at least 2 hours, at least 3, approximately at least 4, or more hours per day for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more days after applying glyphosate to the plant, where the days in which the temperature exceeds the ambient temperature are presented during the vegetative period of the plant.

The following examples are offered by way of illustration and not as a limitation.

Experimental part

Example 1. Design and expression of syngrg 23

GRG23 (SEQ ID NO: 2) is an EPSP synthase that possesses excellent kinetic values for Km, Ki and Vmax (U.S. Patent Application No. 60 / 741,166 filed on December 1, 2005). A new gene sequence that encodes the GRG23 protein was designed and synthesized (US Patent Application No. 60 / 741,166 filed on December 1, 2005). The resulting sequence is supplied here as SEQ ID NO: 6, and referred to herein as "syngrg23". The open reading frame was cloned into the expression vector pRSF1b (Invitrogen) by methods known in the art.

The syngrg23 gene encoding GRG23 was cloned into a pUC19 vector to create pAX748. PCR primers flanking syngrg23 in this vector were used to amplify syngrg23 from pAX748 using the Mutazyme II (Stratagene) system to introduce random mutations in the syngrg23 coding region. Template 1:50 was diluted in the error-prone PCR reaction, and the application was carried out for 30 cycles. The resulting PCR product was digested with the restriction enzymes BamHI and Sgs I, gel purified, and ligated into the vector pRSF1b to create a fine library of syngrg23.

The fine syngrg23 libraries were transformed into E. coli strain BL21 * DE3 star (Invitrogen). After transformation, individual colonies were plated on 1 x M63 medium containing 150 mM glyphosate to select clones that had retained enzyme activity and culture tolerance.

Example 2. Detection of resistance to glyphosate on plates

Library ligaments were transformed into competent E. coli cells of BL21 * DE3 (Invitrogen). The transformations were carried out according to the manufacturer's instructions with the following modifications. After incubation for 1 hour at 37 ° C in SOC medium, the cells were pelleted by centrifugation (5 minutes, 1000 x g, 4 ° C). The cells were washed with 1 ml of M63 +, centrifuged again, and the supernatant was decanted. The cells were washed a second time with 1 ml of M63 + and resuspended in 200 ul of M63 +.

5

For the selection of mutant GRG23 enzymes that confer resistance to glyphosate in E. coli, cells were plated on M63 + agar medium containing 150 mM glyphosate, 0.05 mM IPTG (isopropyl-beta-Diogalactopyranoside), and 50 μg / ml of Kanamycin The M63 + medium contains 100 mM KH2PO4, 15 mM (NH4) 2SO4, 50 μM CaCl2, 1 μM FeSO4, 50 μM MgCl2, 55 mM glucose, 25 mg / liter of L-proline, 10 mg / liter of thiamine hydrochloride,

10 enough NaOH to adjust the pH to 7.0, and 15 g / liter agar. The plates were incubated for 36 hours at 37 ° C.

Individual colonies were selected and arranged in 384 well plates. Two plates of 384 wells were created in this way. A third plate of 384 clones was selected from colonies that grew on plates lacking glyphosate.

fifteen

Example 3. Isolation and analysis of glyphosate resistant GRG23 variants

Transformed BL21 * DE3 cells with mutated variants of syngrg23 and / or grg23 were identified by culturing on glyphosate plates. Extracts of mutated variants of syngrg23 and grgr23 were prepared and analyzed for enhanced enzymatic activity. Identified colonies were immobilized on glyphosate plates in 96-well blocks containing LB medium and cultured to an O.D. about 0.6. IPTG (0.5 mM) was then added and the blocks were incubated overnight at 20 ° C to induce protein expression. Protein extracts were prepared from cell precipitates using POP culture reagent (Novagen) and Lysonase (Novagen), and enzymatic activity was measured in the crude lysates after heating the extracts.

25 for 30 min at 37 ° C. Extracts with an activity greater than two standard deviations above the average of a group of extracts containing the appropriate control protein (eg GRG23) were selected for further analysis.

Clones showing greater activity after incubation as crude extracts were grown in 250

30 mL of LB cultures, and protein expression was induced with IPTG. After induction, the GRG23 mutant protein from each culture was purified by means of affinity chromatography using a cobalt resin (Novagen). The purified proteins were then analyzed for enzymatic activity after heating for 0.2, 4, and approximately 16 hours at 37 ° C.

35 Example 4. Enhanced variants of GRG23.

From a mutated syngrg23 DNA library, different clones with enhanced activity at 37 ° C were identified. The DNA sequences of the clones corresponding to these extracts were determined. Table 1 shows the amino acid changes identified in six variants of GRG23 that retained glyphosate resistance:

40 grg23 (L3P1.B20) (SEQ ID NO: 20) encoding the amino acid sequence GRG23 (L3P1.B20) (SEQ ID NO: 21), grg23 (L3P1.B3) (SEQ ID NO: 22) encoding the amino acid sequence grg23 (L3P1B3) (SEQ ID NO: 23); GRG23 (L3P1F18) (SEQ ID NO: 24) encoding the amino acid sequence GRG23 (L3P1.F18) (SEQ ID NO: 25); and, grg23 (L3P1.O23) (SEQ ID NO: 26) encoding the amino acid sequence GRG23 (L3P1.O23) (SEQ ID NO: 27).

45 Table 1. Mutations identified in glyphosate resistant GRG23 variants

Clone

Amino acid (AA) in GRG23

L3P1B20

V206I

L3P1B3

D75H, E217K

L3P1F18

T274I

L3P1O23

R5H

The clones were grown in 250 mL of LB cultures, and the expression of the induced protein was isolated as described above. The purified proteins were then analyzed for enzymatic activity after

16

heating for 0, 2, 4, and approximately 16 hours at 37 ° C. It was found that one of the clones, called "M5", retained a greater proportion of its enzymatic activity after a prolonged incubation at 37 ° C (Table 2). The DNA sequence of these clones was determined, and the gene was referred to herein as grg23 (ace1) (SEQ ID NO: 8). Protein expressed from grg23 (ace1) was designated as GRG23 (ACE1) (SEQ ID NO: 9).

Table 2. Average life of GRG23 (ACE1) vs. GRG23 at elevated temperature

Protein

Half-life at 37 ° C (hours)

GRG23

7

GRG23 (ACE1)

 15.5

GRG23 (ACE1) contains 2 amino acid substitutions relative to the wild-type GRG23 protein: A49 → T and S276 → T. The pRSFIb vector containing this gene is called pAX3801. Figure 1 shows the relative stability of GRG23 (ACE1) vs. GRG23 at elevated temperatures.

Example 5. Determination of EPSPS activity of variants of GRG-23

Extracts containing proteins of the GRG23 variant were analyzed by the activity of EPSP synthase as described in US Patent Application Number 60 / 741,166 filed on December 1, 2005. Analysis was carried out in a final volume. 50 ul containing 0.5 mM shiquimate-3-phosphate, 200 uM phosphoenolpyruvate (PEP), 1 U / ml xanthine oxidase, 2 U / ml nucleoside phosphorylase, 2.25 mM inosine, 1 U / ml peroxidase of radish, 2 mM glyphosate, 50 mM HEPES / KOH pH 7.0, 100 mM KCl, and AMPLEX® Red (Invitrogen) according to the manufacturer's instructions. Extracts were incubated with all components of the assay except shiquimato-3-phosphate for 5 minutes at room temperature, and the tests were initiated by acquisition of shiquimato-3-phosphate. The activity of EPSP synthase was measured using a Spectramax Gemini XPS fluorescence spectrometer (Molecular Dynamics, excitation: 555 nm; emission: 590 nm).

A complete determination of the kinetic parameters on purified protein was performed as previously described (US Patent Application No. 60 / 741,166 filed on December 1, 2005) by adjusting the amount of protein determined by the Bradford assay. As is known in art. For any glyphosate concentration, the activity of EPSP synthase was measured based on a wide range of PEP concentrations. The data was adjusted to the Michaelis-Menten equation using KALEIDAGRAPH® software (Synergy Software) and was used to determine the Km (apparent Km) of EPSP synthase with that glyphosate concentration. The apparent Km values were determined with no less than four glyphosate concentrations, and the EPSPS Ki for glyphosate was calculated from the apparent Km vs. plot. glyphosate concentration, using the equation (ml * x / (m2 + x); ml = 1; m2 = 1) as known in the art.

Table 3. Kinetics of GRG23 (ACE1) vs. GRG23

Km (uM)

Ki (uM) Vmax (nmol / min / ug)

GRG23

 12.2 13,800 14.77

GRG23 (ACE1)

 9.7 14,620 13.73

Example 6. Identification of grg23 (ace2).

GRG23 (ACE1) contains two amino acid changes relative to GRG23. To determine whether additional substitutions in these positions could further improve activity, a DNA librarian was generated that resulted in clones expressing proteins that were substantially fined at positions 49 and 276 corresponding to GRG23 (SEQ ID NO: 2). Clones that confer resistance to glyphosate were selected by culturing on glyphosate plates, and were cultured and analyzed for the kinetic properties described.

Surprisingly, a clone, referred to herein as grg23 (ace2) (SEQ ID NO: 10), which encodes the GRG23 protein (ACE2) (SEQ ID NO: 11) was identified or have better thermal stability. The DNA sequence of grg23 (ace2) shows that GRG23 (ACE2) contains a single amino acid change (residue 276 of GRG23 for arginine).

Example 7. Comparison of GRG23 and GRG51, and mutagenesis of different residues.

5

Two libraries were generated to evaluate the permutations of possible amino acid sequences from the comparison of the amino acid sequences of GRG23 and GRG51. The first library introduced a variation from the amino acid sequence of GRG51 in a coding region of grg23 (ace2). The second library introduced the variation from the amino acid sequence of GRG23 (ACE2) within the

10 coding region of grg51.

The resulting library clones were evaluated for (1) the ability to confer resistance to glyphosate in a cell, and (2) activity after a prolonged incubation at 37 ° C. A total of clones were sequenced and analyzed in more detail. A particular clone, referred to herein as grg51.4 (SEQ ID NO: 12), that encodes the protein

15 GRG51.4 (SEQ ID NO: 13), contains several amino acid changes in relation to both GRG23 (ACE2) and GRG51. The amino acid changes present in GRG51.4 in relation to GRG23 (ACE2) were subsequently introduced into the grg23 (ace2) gene, to produce grg23 (ace3) (SEQ ID NO: 14), which encodes the GRG23 protein (ACE3) ( SEQ ID NO: 15). GRG23 (ACE3) exhibits superior activity and thermal stability in relation to GRG23, and GRG23 (ACE2).

GRG23 was mutated (ace1), and the clones were analyzed to identify clones expressing variants with improved thermal stability and / or activity. One clone, grg23 (L5P2.J2) (SEQ ID NO: 16), which encodes GRG23 (L5P2.J2) (SEQ ID NO: 17), was identified by virtue of its improved kinetic properties. GRG23 (L5P2.J2) contains three amino acid changes relative to GRG23 (ACE1), as shown in the following Table 4.

25

Table 4. Amino acid changes in GRG23 (L5P2.J2)

Amino acid (AA) in GRG23 (L5P2.J2) in relation to GRG23 (ACE1)

V101F

A213S

D284N

Oligonucleotide mutagenesis was used to modify the coding region of grg23 (ace3) to contain

30 each of the amino acid changes identified in GRG23 (L5P2.J2). A clone was identified with a gene that contains these modifications, and it was found to encode a protein that has altered kinetic properties on GRG23 (ACE3). This gene was called grg23 (ace4) (SEQ ID NO: 18). The protein encoded by grg23 (ace4) and designated as GRG23 (ACE4) (SEQ ID NO: 19) contains a single amino acid change relative to GRG23 (ACE3) (Valine 101 for phenylalanine). Based on this result, mutagenesis was carried out

35 of a separate oligonucleotide to analyze the kinetics of each possible amino acid substitution at the position

101. None of the amino acid changes resulted in a further improvement in kinetic properties compared to GRG23 (ACE4). See Table 5.

Table 5. Kinetics of improved variants

40

 Km (μM)

Ki (μM) Vmax (nmol / min / μg)

GRG23

 14 10,800 13

GRG51

 fifteen 21,048 13

GRG23 (ACE1)

 10 14,620 14

GRG23 (ACE2)

 eleven 18,104 fifteen

GRG51.4

 19 26,610 17

GRG23 (ACE3)

 fifteen 20,000 17

GRG23 (L5P2.J2)

fifteen 2,500 2. 3

GRG23 (ACE4)

 14 5,010 24

Example 8. Improved thermal stability of variants of GRG23

The thermal stability of GRG23 and different variants was determined by incubating protein samples at 37 ° C for a range of times, then determining the residual activity of EPSPS as described herein, and comparing the activity with that of a sample. of control incubated at 4 ° C. Table 6 shows that the variants of GRG23, GRG23 (ACE1), GRG23 (ACE2), and GRG23 (ACE3) have better thermal stability.

Table 6. Thermal stability of the GRG23 and GRG23 variants

Protein

 t1 / 2 at 37 ° C

GRG23

10.1

GRG23 (ACE1)

 15.3

GRG23 (ACE2)

 34.2

GRG23 (ACE3)

 65.4

10

Example 9. Variants of grg23 (ace3)

Oligonucleotide mutagenesis was used to generate variants of grg23 (ace3) resulting in the expression of proteins with modifications for amino acid residues corresponding to positions 169 to 174 of SEQ 15 ID NO: 15. Mutagenesis reactions were carried out. using the QuickChange® (Stratagene) kit according to the manufacturer's instructions. Plasmid clones were transformed into the E. coli BL21 * DE3 cell line, and protein expression was induced by IPTG as is known in the art. Proteins were purified by means of affinity binding with a nickel column (TALON metal affinity resin, Clontech). The native protein GRG23 (ace3) was also expressed and purified for use as

20 control After purification of each enzyme, the protein concentration was measured by the Bradford assay as is known in the art.

Example 10. Kinetic analysis of the variants of GRG23 (ace3)

The variants of GRG23 (ace3), GRG23 (ace3) R173K (SEQ ID NO: 29, encoded by SEQ ID NO: 28), GRG23 (ace3) G169V / L170V (SEQ ID NO: 31, encoded by SEQ ID NO: 30), GRG23 (ace3) R173Q (SEQ ID NO: 33, encoded by SEQ ID NO: 32), and GRG23 (ace3) I174V (SEQ ID NO: 35, encoded by SEQ ID NO: 34) were characterized by enzymatic assays as described herein, and compared with the native enzyme GRG23 (ace3). For each enzyme, the Km (apparent) was determined for each of the different concentrations of

30 glyphosate, and a graph of Km (apparent) vs. Glyphosate concentration to calculate the Ki for each enzyme. The thermal stability for each enzyme was evaluated by incubation of the enzyme at 37 ° C for 16 hours, and then the remaining enzyme activity (as Vmax) was quantified. the control enzyme that was incubated at 4 ° C.

Kinetic analysis reveals that GRG23 (ace3) R173K, GRG23 (ace3) R173Q, GRG23 (ace3) I174V and GRG23

35 (ace3) G169V / L170V are virtually indistinguishable from native GRG23 (ace3) and show almost identical catalytic velocity, extremely high resistance to glyphosate, and very good PEP affinity. The Km observed for PEP of 19 μM for G169V / L170V (vs. 15 μM for GRG23 (ace3) may represent a slightly lower binding affinity for PEP in relation to GRG23 (ace3). Each of the four variants had a thermal stability greater than 95% after 16 hours at 37 ° C. The kinetic values determined for the

40 variants of GRG23 (ace3), GRG23 (ace3) R173K, GRG23 (ace3) R173Q, and GRG23 (ace3) I174V are shown in Table 6.

Table 7. Kinetics of variants of GRG23 (ace3)

 Km (μM)

Ki (mM) Vmax (nmol / min / μg)

GRG23 (ace3)

 16 14 16

GRG23 (ace3) R173K

16.6 14.7 17.7

GRG23 (ace3) R173Q

14.3 14.2 15.8

GRG23 (ace3) 1174V

15.5 15.0 15.5

Example 11. Cloning of the syngrg23 and grg23 variants in a plant expression cassette.

5

For syngrg23, and each of the variants of grg23 described here (including, for example, grg23 (ace1), grg23 (ace2), grg23 (ace3), grg23 (ace4), grg23 (ace3) R173K, grg23 (ace3) R173Q , grg23 (ace3) I174V, grg23 (ace3) G169V / L170V and grg23 (L5P2.J2)), the open reading frame (ORF) is amplified by means of the PCR of a full-length DNA template. Hind III restriction sites are added to each end of the ORFs during PCR.

Additionally, the ACC 5 nucleotide sequence is added immediately to the start codon of the gene to increase translation efficiency (Kozak (1987) Nucleic Acids Research 15: 8125-8148; Joshi (1987) Nucleic Acids Research 15: 6643 - 6653). The PCR product is cloned and sequenced using techniques well known in the art to ensure that no mutations are introduced during the PCR.

The plasmid containing the PCR product is digested with Hind III and the fragment containing the intact ORF is isolated. This fragment is cloned into the Hind III site of a plasmid such as pAX200, an expression vector of a plant containing the rice actin promoter (McElroy et al. (1991) Molec. Gen. Genet. 231: 150- 160) and the PinII terminator (An et al. (1989) The Plant Cell 1: 115-122). The promoter-gene-terminator fragment of this intermediate plasmid is then subcloned into plasmid pSB11 (Japan Tobacco, Inc.) to form a plasmid.

20 final based on pSB11. In some cases, it may be preferable to generate an alternate construct in which a chloroplast leader sequence is encoded as a fusion with the N terminal of the syngrg23, grg23 (ace1), grg23 (ace2), grg23 (ace3), grg23 ( ace4), grg23 (L5P2.J2), grg23 (ace3) R173K, grg23 (ace3) R173Q, grg23 (ace3) I174V, or grg23 (ace3) G169V / L170V. These pSB11-based plasmids are typically organized such that the DNA fragment containing the promoter-gene-terminator construct, or the construct

The leading chloroplast-terminator promoter-gene can be cut by double digestion by restriction enzymes, such as Kpn I and Pme I, and used for transformation into plants by injection of an aerosol beam. The structure of the resulting clones based on pSB11 is verified by restriction digestion and gel electrophoresis, and by sequencing through the different cloning junctions.

30 The plasmid is mobilized within the LBA4404 strain of Agrobacterium tumefaciens that also houses plasmid pSB1 (Japan Tobacco, Inc.), using triparental conjugation procedures well known in the art, and plating on medium containing spectinomycin. The plasmid clone based on pSB11 has resistance to spectinomycin but is a narrow range host plasmid and cannot replicate in

35 Agrobacterium. Spectinomycin-resistant colonies arise when plasmids based on pSB 11 are integrated into the broad-range plasmid as a pSB1 host through homologous recombination. The jointly integrated product of pSB1 and the plasmid based on pSB11 is verified by Southern hybridization. The Agrobacterium strain that houses the integrated jointly is used to transform corn by means of methods known in the art, such as, for example, the PureIntro method (Japan Tobacco).

40

Example 12. Transformation of plant cells by means of Agrobacterium-mediated transformation

Corn ears are preferably collected 8-12 days after pollination. The embryos are isolated from the spikes, and those embryos with a size of 0.8-1.5 mm are preferred for use in the transformation. Embryos are plated on the plate with the gusset up on a suitable incubation medium, such as DN62A5S medium (3.98 g / L of Sales N6; 1 ml / L (of 1000x Standard) of Vitamins N6; 800 mg / L of L-Asparagine; 100 mg / L of Myo-inositol; 1.4 g / L of L-Proline; 100 mg / L of Casamino acids; 50 g / L of sucrose; 1 ml / L (of a standard 1 mg / ml) 2,4-D). However, suitable media and salts are known in the art and

other than DN62A5S. Embryos are incubated overnight at 25 ° C in the dark. However, it is not really necessary to incubate the embryos overnight.

The explants are transferred to square meshes (30-40 per plate), transferred onto osmotic medium 5 for approximately 30-45 minutes, then transferred to a radiant plate (see, for example, PCT Publication No. WO / 0138514 and the Patent of the United States No. 5,240,842).

The DNA constructs designed to express the GRG proteins of the present invention in plant cells are accelerated within the plant tissue using an aerosol beam accelerator, using 10 conditions essentially as described in PCT Publication No. WO / 0138514. After irradiation, the embryos are incubated for approximately 30 min on an osmotic medium, and placed on incubation medium overnight at 25 ° C in the dark. To avoid unduly damaging irradiated explants, if you incubate them for at least 24 hours before transferring them to a recovery medium. The embryos are then scattered on a medium during the recovery period, approximately for 5 days, 25 ° C in the dark, then transferred to a selection medium. The explants are incubated in a selection medium for up to eight weeks, depending on the nature and characteristics of the particular selection used. After the selection period, the resulting callus is transferred to the embryo maturation medium, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under a dim light, and the regeneration process is initiated by methods known in the art. It is allowed that

20 resulting shoots form roots on rooting medium, and the resulting plants are transferred to pots and propagated as transgenic plants.

materials

25 Medium DN62A5S

Components

 per liter Source

Chu Basal Salt Mix N6 (Prod. No. C 416)

3.98 g / L Phytotechnology Labs

Chu Vitamin N6 Solution (Prod. No. C 149)

1ml / L (from 1000x Stock) Phytotechnology Labs

L-Asparagine

 800 mg / L Phytotechnology Labs

Myo-inositol

 100mg / L Sigma

L-Proline

 1.4 g / L Phytotechnology Labs

Casamino Acids

100 mg / L Fisher Scientific

Saccharose

 50 g / L Phytotechnology Labs

2, 4-D (Prod. No. D-7299)

1m / L (from 1 mg / ml Standard) Sigma

Adjust the pH of the solution to pH 5.8 with 1 N KOH / 1 N KCl, add Gelrite (Sigma) to 3 g / L, and autoclave. After cooling to 50 ° C, add 2 ml / L of a 5 mg / ml standard solution of Silver Nitrate

30 (Phytotechnology Labs). The recipe produces approximately 20 plates.

Example 13. Transformation of EPSP synthase enzymes in Corn Plant Cells through Agrobacterium-mediated transformation

35 The spikes are best collected 8-12 days after pollination. The embryos are isolated from the spikes, and those embryos with a size of 0.8-1.5 mm are preferred for use in the transformation. Embryos are plated on the plate with the skull upward on a suitable incubation medium, and incubated overnight at 25 ° C in the dark.

40 However, it is not really necessary to incubate the embryos overnight. Embryos are contacted with an Agrobacterium strain containing the appropriate vectors that have an EPSP synthase enzyme of the present invention for transfer mediated by the Ti plasmid for approximately 5-10 min, and then plated on a medium of joint culture for approximately 3 days (25 ° C in the dark). After the whole culture, the explants are transferred to a medium during the recovery period

45 approximately for five days (at 25 ° C in the dark). The explants are incubated in the selection medium for up to eight weeks, depending on the nature and characteristics of the particular selection used. After the selection period, the resulting callus is transferred to an embryo maturation medium, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under a dim light, and the regeneration process begins as is known in the art. The resulting shoots are allowed to form roots on a rooting medium, and the resulting plants are transferred to pots and propagated as transgenic plants.

SEQUENCE LISTINGS

<110> Laura C. Schouten Cheryl L. Peters Brian Vande Berg Todd K. Hinson

<120> GRG23 EPSP IMPROVED SYNTHESES: COMPOSITIONS AND METHODS OF USE

<130> 45600/336802

<150> 60 / 972.502

<151> 2007-09-14

<150> 60 / 872.200

<151> 2006-12-01

<150> 60 / 861,455

<151> 2006-11-29

<160> 35

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 1892

<212> DNA

<213> Arthrobacter globiformis

<220>

<221> CDS

<222> (178) ... (1417)

<221> new feature

<222> 1801

<223> n = A, T, C or G

<400> 1

image 1

image 1

image 1

<210> 2

<211> 413

<212> PRT

<213> Arthrobacter globiformis

<400> 2

image 1

image 1

<210> 3

<211> 436

<212> PRT

<213> Arthrobacter globiformis

<400> 3

image 1

<210> 4

<211> 1242

<212> DNA 5 <213> iUnknown

<220>

<223> isolated from soil sample

<221> CDS

<222> (1) ... (1242) 10 <400> 4

image 1

image 1

image2

<210> 5

<213> iUnknown

<220>

<223> isolated from a soil sample

<400> 5

10

image 1

<210> 6

<211> 1242

<212> DNA

<213> Artificial Sequence

<220>

<223> grg23 synthetic

<221> CDS

<222> (1) ... (1242)

<400> 6

image 1

image 1

image2

<210> 7

<213> Artificial Sequence

<220>

<223> synthetic GRG23

<400> 7

10

image 1

image 1

<210> 8

<211> 1242 5 <212> DNA

<213> Artificial Sequence

<220>

<223> grg23 (ace1)

<221> CDS 10 <222> (1) ... (1292)

<400> 8

image 1

image 1

image2

<210> 9

<213> Artificial Sequence

<220>

<223> GRG23 (ACE1)

<400> 9

10

image 1

image 1

<210> 10

<211> 1242

<212> DNA 5 <213> Artificial Sequence

<220>

<223> grg23 (ace2)

<221> CDS

<222> (1) ... (1242) 10 <400> 10

image 1

image 1

image 1

<210> 11

<211> 413

<212> PRT

<213> Artificial Sequence

<220>

<223> GRG23 (ACE2)

<400> 11

image 1

image 1

<210> 12

<211> 1242 5 <212> DNA

<213> Artificial Sequence

<220>

<223> grg51.4

<221> CDS 10 <222> (1) ... (1242)

<400> 12

image 1

image 1

image2

<210> 13

<213> Artificial Sequence

<220>

<223> GRG51.4

<400> 13

10

image 1

image 1

<210> 14

<211> 1242

<212> DNA 5 <213> Artificial Sequence

<220>

<223> grg23 (ace3)

<221> CDS

<222> (1) ... (1242) 10 <400> 14

image 1

image 1

image2

<210> 15

<213> Artificial Sequence

<220>

<223> GRG23 (ACE3)

<400> 15

10

image 1

image 1

<210> 16

<211> 1242

<212> DNA 5 <213> Artificial Sequence

<220>

<223> grg23 (L5P2.J2)

<221> CDS

<222> (1) ... (1242) 10 <400> 16

image 1

image 1

image2

<210> 17

<213> Artificial Sequence

<220>

<223> GRG23 (L5P2.J2)

<400> 17

10

image 1

image 1

<210> 18

<211> 1242 5 <212> DNA

<213> Artificial Sequence

<220>

<223> grg23 (ace4)

<221> CDS 10 <222> (1) ... (1292)

<400> 18

image 1

image 1

image2

<210> 19

<213> Artificial Sequence

<220>

<223> GRG23 (ACE4)

<400> 19

10

image 1

image 1

<210> 20

<211> 1244

<212> DNA 5 <213> Artificial Sequence

<220>

<223> grg23 (L3P1.B20)

<221> CDS

<222> (1) ... (1242) 10 <400> 20

image 1

image 1

image 1

<210> 21

<211> 413

<212> PRT

<213> Artificial Sequence

<220>

<223> GRG23 (L3P1.B20)

<400> 21

image 1

image 1

<210> 22

<211> 1244 5 <212> DNA

<213> Artificial Sequence

<220>

<223> grg23 (L3Pl.B3)

<221> CDS 10 <222> (1) ... (1242)

<400> 22

image 1

image 1

image 1

<210> 23

<211> 413

<212> PRT

<213> Artificial Sequence

<220>

<223> GRG23 (L3P1.B3)

<400> 23

image 1

image 1

<210> 24

<211> 1244

<212> DNA 5 <213> Artificial Sequence

<220>

<223> grg23 (L3P1.F18)

<221> CDS

<222> (1) ... (1242) 10 <400> 24

image 1

image 1

image 1

<210> 25

<211> 413

<212> PRT

<213> Artificial Sequence

<220>

<223> GRG23 (L3P1.F18)

<400> 25

image 1

image 1

<210> 26

<211> 1244 5 <212> DNA

<213> Artificial Sequence

<220>

<223> grg23 (L3P1.023)

<221> CDS 10 <222> (1) ... (1242)

<400> 26

image 1

image 1

image2

<210> 27

<213> Artificial Sequence

<220>

<223> GRG23 (L3P1.023)

<400> 27

10

image 1

image 1

<210> 28

<211> 1244 5 <212> DNA

<213> Artificial Sequence

<220>

<223> sequence of a variant of grg23 (ace3), (grg23 (ace3) R173K)

<221> CDS 10 <222> (1) ... (1242)

<400> 28

image 1

image 1

image 1

<210> 29

<211> 413

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of a variant of GRG23 (ace3), (GRG23 (ace3) R173K)

<400> 29 <210> 30

image 1

image 1

<211> 1244

<212> DNA 5 <213> Artificial Sequence

<220>

<223> sequence of a variant of grg23 (ace3), (grg23 (ace3) G169V / L170V)

<221> CDS

<222> (1) ... (1242) 10 <400> 30

image 1

image 1

image 1

<210> 31

<211> 413

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of a variant of GRG23 (ace3), (GRG23 (ace3) G169V / L170V)

<400> 31 <210> 32

image 1

image 1

<211> 1244 5 <212> DNA

<213> Artificial Sequence

<220>

<223> sequence of a variant of grg23 (ace3), (grg23 (ace3) R173Q)

<221> CDS 10 <222> (1) ... (1292)

<400> 32

image 1

image 1

image 1

<210> 33

<211> 413

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of a variant of GRG23 (ace3), (GRG23 (ace3) R173Q)

<400> 33 <210> 34

image 1

image 1

<211> 1244 5 <212> DNA

<213> Artificial Sequence

<220>

<223> sequence of a variant of grg23 (ace3), (grg23 (ace3) 1174V)

<221> CDS 10 <222> (1) ... (1242)

<400> 34

image 1

image 1

image 1

<210> 35

<211> 413

<212> PRT

<213> Artificial Sequence

<220>

<223> sequence of a variant of GRG23 (ace3), (GRG23 (ace3) I174V)

<400> 35

image 1

image 1

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is solely for the convenience of the reader. It is not part of the European patent document. Although great care has been taken in the collection, errors or omissions cannot be excluded and the EPO rejects any responsibility in this regard.

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US 4769061 A [0004] [0005] • WO 9706269 A [0044]

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US 5094945 A [0004] [0005] • WO 9514098 A [0044]

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WO 9741228 A [0043] • WO 0138514 A [0105] [0106]

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US 4771002 A [0043] • US 5240842 A [0105]

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Harlow; Lane Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, 1988 [0039]

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Roberts et al. Proc. Natl Acad. Sci. USA, 1979, vol. 76, 760-764 [0042]

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Claims (16)

one.

 A nucleic acid molecule selected from the group consisting of:

a) a nucleic acid molecule containing the nucleotide sequence of SEQ ID NOs: 28, 30, 32, or 34; and, b) a nucleic acid molecule encoding a polypeptide containing the amino acid sequence of SEQ ID NOs: 29, 31, 33, or 35.

2.

 The nucleic acid molecule of claim 1, wherein said nucleotide sequence is operably linked to a promoter capable of directing the expression of a coding sequence in a plant cell.

3.

 The nucleic acid molecule of claim 1 or 2, wherein said nucleotide sequence is a synthetic sequence that has been designed for expression in a plant.

Four.

 A vector containing the nucleic acid molecule of claim 1, 2, or 3, preferably additionally containing a nucleic acid molecule encoding a heterologous polypeptide.

5.

 A host cell containing the nucleic acid molecule of claim 2 or 3, preferably that is a bacterial host cell or a plant cell.

6.

 A transgenic plant containing the host cell of the plant of claim 5, preferably wherein said plant is selected from the group consisting of corn, sorghum, wheat, sunflower, tomato, cruciferous, peppers, potato, cotton, rice, soybeans, Sugar beet, sugar cane, tobacco, barley, and oilseed rape.

7.

 A transgenic seed containing the nucleic acid molecule of claim 1, 2, or 3.

8.

 A recombinant polypeptide selected from the group consisting of:

a) a polypeptide containing the amino acid sequence of SEQ ID NOs: 29, 31, 33, or 35; and, b) a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 28, 30, 32, or 34, preferably additionally containing a heterologous amino acid sequence.

9.

 A method of producing a polypeptide with herbicide resistance activity, comprising culturing the host cell of claim 5 under conditions in which a nucleic acid molecule encoding the polypeptide is expressed, said polypeptide being selected from the group that it consists of:

a) a polypeptide containing the amino acid sequence of SEQ ID NOs: 29, 31, 33, or 35; and, b) a polypeptide encoded by the nucleotide sequence of SEQ ID NOs: 28, 30, 32, or 34.

10.

 A method for conferring resistance to a herbicide in a plant, said method comprising transforming said plant with a DNA construct, said construction comprising a promoter that directs expression in a cell of a plant operatively linked to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 28, 30, 32, or 34, and the regeneration of a transformed plant, preferably wherein said herbicide is glyphosate.

eleven.

 A plant that has stably incorporated into its genome a DNA construct that contains a nucleotide sequence that encodes a protein that has herbicide resistance activity, wherein said nucleotide sequence is selected from the group consisting of:

a) a nucleic acid molecule containing the nucleotide sequence of SEQ ID NOs: 28, 30, 32, or 34; and, b) a nucleic acid molecule encoding a polypeptide containing the amino acid sequence of SEQ ID NOs: 29, 31, 33, or 35; wherein said nucleotide sequence is operatively linked to a promoter that directs the expression of a coding sequence in a plant cell.

12.

 The plant of claim 11, wherein said plant is a cell of a plant, a soybean plant, or a Corn plant.

13.

 The plant of claim 11, wherein said plant is selected from the group consisting of corn, sorghum, wheat, sunflower, tomato, cruciferous, peppers, potato, cotton, rice, soy, sugar beet, sugar cane, tobacco, barley, and oilseed rapeseed.

14.

 A method to increase vigor or productivity in a plant comprising a product: a) provide a plant containing the nucleotide sequence set forth in SEQ ID NOs: 28, 30, 32, or 34; b) contacting said plant with an effective glyphosate concentration; Y, c) cultivate said plant under conditions where the temperature is higher than the ambient temperature at least for two consecutive hours per day for at least four days after contact with said glyphosate, where these days after the contact are within the vegetative period of the plant,

wherein the vigor or productivity of said plant are greater than the vigor or productivity of a plant that expresses an EPSP synthase of glyphosate tolerance that does not have an optimum temperature higher than the temperature of the environment, preferably where the temperature in step (c) is about 32 ° C to about 60 ° C.

fifteen.

 A method for conferring glyphosate resistance in a plant comprising:

a) provide a plant containing the nucleotide sequence set forth in SEQ ID NOs: 28, 30, 32, or 34; b) contacting said plant with an effective glyphosate concentration; Y, c) cultivate said plant under conditions where the temperature is higher than the ambient temperature at least for two consecutive hours per day for at least four days after contact with said glyphosate, where these days after the contact are within the vegetative period of the plant.

16.

 The method of claim 14, wherein the temperature in step (c) is about 32 ° C to about 60 ° C, preferably wherein the temperature in step (b) is about 37 ° C.