# Patent

## Title
Identification of a new class of EPSP synthases

## Abstract

Compositions and methods for conferring tolerance to glyphosate in bacteria, plants, plant cells, tissues and seeds are provided. Compositions include a novel class of EPSPS enzymes, designated Class III, and polynucleotides encoding such enzymes, vectors comprising those polynucleotides, and host cells comprising the vectors. The novel proteins comprise at least one sequence domain selected from the Class III domains provided herein. These sequence domains can be used to identify EPSP synthases with glyphosate resistance activity.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application Ser. Nos. 60/669,686, filed Apr. 8, 2005; 60/678,348 filed May 6, 2005; 60/695,193 filed Jun. 29, 2005; and 60/725,182 filed Oct. 11, 2005, the contents of which are hereby incorporated in their entirety by reference herein.


FIELD OF THE INVENTION
This invention relates to plant molecular biology, particularly to a novel class of EPSP synthases that confer resistance to the herbicide glyphosate.
BACKGROUND OF THE INVENTION
N-phosphonomethylglycine, commonly referred to as glyphosate, is an important agronomic chemical. Glyphosate inhibits the enzyme that converts phosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid (S3P) to 5-enolpyruvyl-3-phosphoshikimic acid. Inhibition of this enzyme (5-enolpyruvylshikimate-3-phosphate synthase; referred to herein as “EPSP synthase”, or “EPSPS”) kills plant cells by shutting down the shikimate pathway, thereby inhibiting aromatic amino acid biosynthesis.
Since glyphosate-class herbicides inhibit aromatic amino acid biosynthesis, they not only kill plant cells, but are also toxic to bacterial cells. Glyphosate inhibits many bacterial EPSP synthases, and thus is toxic to these bacteria. However, certain bacterial EPSP 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 from Agrobacterium tumefaciens strain CP4 has been used to confer herbicide resistance on plant cells following expression in plants. A mutated EPSP synthase from Salmonella typhimurium strain CT7 confers glyphosate resistance in bacterial cells, and confers glyphosate resistance on plant cells (U.S. Pat. Nos. 4,535,060; 4,769,061; and 5,094,945).
EPSP synthase (Mr 46,000) folds into two similar domains, each comprising three copies of a βαβαββ-folding unit (Stallings et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 885046-5050). Lys-22, Arg-124, Asp-313, Arg-344, Arg-386, and Lys-411 are conserved residues of the EPSP synthase from E. coli (Schönbrunn et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:1376-1380). Conserved residues important for EPSPS activity also include Arg-100, Asp-242, and Asp-384 (Selvapandiyan et al. (1995) FEBS Letters 374:253-256). Arg-27 binds to S3P (Shuttleworth et al. (1999) Biochemistry 38:296-302). Variants of the wild-type EPSPS enzyme have been isolated which are glyphosate-tolerant as a result of alterations in the EPSPS amino acid coding sequence (Kishore and Shah (1988) Annu. Rev. Biochem. 57:627-63; Wang et al. (2003) J. Plant Res. 116:455-60; Eschenburg et al. (2002) Planta 216:129-35). He et al. (2001, Biochim et Biophysica Acta 1568:1-6) have developed EPSP synthases with increased glyphosate tolerance by mutagenesis and recombination between the E. coli and Salmonella typhimurium EPSPS genes, and suggest that mutations at position 42 (T42M) and position 230 (Q230K) are likely responsible for the observed resistance. Subsequent work (He et al (2003) Biosci. Biotech. Biochem. 67:1405-1409) shows that the T42M mutation (threonine to methionine) is sufficient to improve tolerance of both the E.coli and Salmonella typhimurium enzymes.
Due to the many advantages herbicide resistance plants provide, methods for identifying herbicide resistance genes with glyphosate resistance activity are desirable.
SUMMARY OF INVENTION
Compositions and methods for conferring tolerance to glyphosate in bacteria, plants, plant cells, tissues and seeds are provided. Compositions include a novel class of EPSPS enzymes, designated Class III, and polynucleotides encoding such enzymes, vectors comprising those polynucleotides, and host cells comprising the vectors. The novel proteins comprise at least one sequence domain selected from the following domains (Class III domains):
Domain I:
L-A-K-G-X1-S-X2-L-X3-G-A-L-K-S-D-D-T (SEQ ID NO: 13), where X1 denotes lysine or threonine, where X2 denotes arginine or histidine, where X3 denotes serine or threonine;
Domain Ia:
L-A-K-G-X1, (SEQ ID No:14), where X1 denotes lysine or threonine;
Domain Ib:
S-X1-L-X2 (SEQ ID NO: 15), where X1 denotes arginine or histidine, where X2 denotes serine or threonine;
Domain Ic:











G-A-L-K-S-D-D-T;
(SEQ ID NO: 16)







Domain II:

E-P-D-X1-X2-T-F-X3-V-X4-X5-X6-G (SEQ ID NO:17), where X1 denotes aspartic acid or alanine, where X2 denotes serine or threonine, where X3 denotes valine or isoleucine, where X4 denotes threonine or glutamic acid or lysine, where X5 denotes serine or glycine, where X6 denotes glutamine or serine or glutamic acid or threonine;

Domain IIa:

E-P-D-X1-X2-T-F-X3-V (SEQ ID NO: 18), where X1 denotes aspartic acid or alanine, where X2 denotes serine or threonine, where X3 denotes valine or isoleucine;

Domain IIb:

X1-X2-X3-G (SEQ ID NO:19), where X1 denotes threonine or glutamic acid or lysine, where X2 denotes serine or glycine, where X3 denotes glutamine or serine or glutamic acid or threonine;

Domain III:












RFLTAA;
(SEQ ID NO: 20)







Domain IV:












KRPI(G/M)P
(SEQ ID NO: 21)







K-R-P-I-X1-P, where X1 denotes glycine or methionine or leucine;

Domain V:

X1-G-C-P-P-V (SEQ ID NO:22), where X1 denotes threonine or serine;

Domain VI:

I-G-A-X1-G-Y-X2-D-L-T (SEQ ID NO:23), where X1 denotes arginine or lysine or leucine, and where X2 denotes isoleucine or valine;

Domain VII:

W-X1-V-X2-X3-T-G (SEQ ID NO:24), where X1 denotes arginine or lysine, where X2 denotes alanine or histidine or glutamic acid or serine, where X3 denotes proline or alanine;

Domain VIII:

E-P-D-A-S-A-A-T-Y-L-W-X1-A-X2-X3-L (SEQ ID NO:25), where X1 denotes alanine or glycine, where X2 denotes glutamic acid or glutamine, where X3 denotes valine or leucine or alanine;

Domain VIIIa:












E-P-D-A-S-A-A-T-Y-L-W;
(SEQ ID NO: 26)







Domain IX:

I-D-X1-G (SEQ ID NO:27), where X1 denotes isoleucine or leucine;

Domain X:

F-X1-Q-P-D-A-K-A (SEQ ID NO:28), where X1 denotes threonine or serine;

Domain XI:

X1-F-P-X2-X3-X4-A-X5-X6-X7-G-S-Q-M-Q-D-A-I-P-T-X8-A-V-X9A-A-F-N (SEQ ID NO:29), where X1 denotes glutamine or lysine or serine, where X2 denotes asparagine or histidine, where X3 denotes methionine or leucine, where X4 denotes proline or glutamine, where X5 denotes threonine or glutamic acid or valine, where X6 denotes valine or isoleucine, where X7 denotes aspartic acid or valine, where X8 denotes leucine or isoleucine, where X9 denotes leucine or isoleucine;

Domain XIa:

X1-F-P-X2-X3-X4-A (SEQ ID NO:30), where X1 denotes glutamine or lysine or serine, where X2 denotes asparagine or histidine, where X3 denotes methionine or leucine, where X4 denotes proline or glutamine;

Domain XIb:

X1-X2-X3-G-S-Q-M-Q-D-A-I-P-T-X4-A-V-X5A-A-F-N (SEQ ID NO:31), where X1 denotes threonine or glutamic acid or valine, where X2 denotes valine or isoleucine, where X3 denotes aspartic acid or valine, where X4 denotes leucine or isoleucine, where X5 denotes leucine or isoleucine;

Domain XIc:












G-S-Q-M-Q-D-A-I-P-T;
(SEQ ID NO: 32)







Domain XII:

P-V-R-F-X1-X2-X3-X4-N-L-R-V-K-E-C-D-R-X5 (SEQ ID NO:33), where X1 denotes valine or threonine, where X2 denotes glutamic acid or glycine, where X3 denotes leucine or isoleucine, where X4 denotes alanine or glutamic acid, where X5 denotes isoleucine or valine;

Domain XIIa:












P-V-R-F;
(SEQ ID NO: 34)







Domain XIIb:

X1-X2-X3-X4-N-L-R-V-K-E-C-D-R-X5 (SEQ ID NO:35), where X1 denotes valine or threonine, where X2 denotes glutamic acid or glycine, where X3 denotes leucine or isoleucine, where X4 denotes alanine or glutamic acid, where X5 denotes isoleucine or valine;

Domain XIIc:












N-L-R-V-K-E-C-D-R;
(SEQ ID NO: 36)







Domain XIII:

E-G-D-D-L-X1-X2 (SEQ ID NO:37), where X1 denotes leucine or isoleucine, where X2 denotes valine or isoleucine;

Domain XIV:

X1-P-X2-L-A-G (SEQ ID NO:38), where X1 denotes aspartic acid or asparagine, where X2 denotes alanine or serine or threonine;

Domain XV:

A-X1-I-D-X2-X3-X4-D-H-R (SEQ ID NO:39), where X1 denotes leucine or serine or glutamic acid, where X2 denotes threonine or serine, where X3 denotes histidine or phenylalanine, where X4 denotes alanine or serine;

Domain XVI:

F-A-L-A-X1-L-K-X2-X3-G-I (SEQ ID NO:40), where X1 denotes glycine or alanine, where X2 denotes isoleucine or valine, where X3 denotes serine or glycine or alanine or lysine;

Domain XVIa:

F-A-L-A-X1-L-K (SEQ ID NO:41), where X1 denotes glycine or alanine;

Domain XVIb:

L-K-X1-X2-G-I (SEQ ID NO:42), where X1 denotes isoleucine or valine, where X2 denotes serine or glycine or alanine or lysine; and

Domain XVII:

-X1-P-X2-C-V-X3-K (SEQ ID NO:43), where X1 denotes asparagine or aspartic acid, where X2 denotes alanine or aspartic acid, where X3 denotes alanine or glycine.

Domain XVIII:

X1-S-L-G-V (SEQ ID NO:44), where X1 denotes alanine or serine or proline.

The above domains set forth in SEQ ID NOS:13-44 were identified by aligning Class III sequences that share at least 50% sequence identity. The presence of at least one of these sequence domains is predictive of glyphosate resistance activity.
Isolated nucleic acid molecules corresponding to herbicide resistance-conferring nucleic acid sequences are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising a Class III domain, including the nucleotide sequence set forth in SEQ ID NOS:9, 11, 55, 57 and 58, a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:10, 12, 56, and 59, the herbicide resistance nucleotide sequence deposited in a bacterial host as Accession Nos. B-30833 and B-30838, as well as variants and fragments thereof. Nucleotide sequences that are complementary to a nucleotide sequence of the invention, or that hybridize to a sequence of the invention are also encompassed. The sequences find use in the construction of expression vectors for subsequent transformation into plants of interest, as probes for the isolation of other glyphosate resistance genes, as selectable markers, and the like.
Plasmids containing the herbicide resistance nucleotide sequences of the invention were deposited in the permanent collection of the Agricultural Research Service Culture Collection, Northern Regional Research Laboratory (NRRL) on Apr. 4, 2005, and assigned Accession No. B-30833. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Access to these deposits will be available during the pendency of the application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. Upon allowance of any claims in the application, the Applicants will make available to the public, pursuant to 37 C.F.R. §1.808, sample(s) of the deposit with the ATCC. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Compositions also include antibodies to the polypeptides as well as synthetic polynucleotides encoding herbicide resistance polypeptides. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds that are glyphosate tolerant by the introduction of the compositions of the invention into the genome of the organism. Where the organism is a plant, the introduction of the sequence allows for glyphosate containing herbicides to be applied to the crop to selectively kill the glyphosate sensitive weeds, but not the transformed organism.
Methods for identifying an EPSP synthase with glyphosate resistance activity are additionally provided. The methods comprise obtaining an amino acid sequence for an EPSP synthase, and identifying whether the amino acid sequence comprises at least one sequence domain of the invention.
The EPSP synthases described herein represent a new class of EPSPS enzymes, referred to hereinafter as Class III EPSPS enzymes.



DESCRIPTION OF FIGURES
FIG. 1A-C shows an alignment of EPSP synthase amino acid sequences. The conserved residues that have been identified as important for substrate binding and for EPSPS activity are boxed. The boxes delineate the amino acid locations of the Class III domains. Roman numerals above the boxes correspond to the Class III domains.



DETAILED DESCRIPTION OF THE INVENTION
The present invention is drawn to compositions and methods for conferring herbicide tolerance, particularly glyphosate tolerance, in organisms is provided. The methods involve transforming organisms with nucleotide sequences encoding a Class III glyphosate tolerance gene of the invention. In particular, the present invention recognizes a class of enzymes that confers glyphosate tolerance and nucleotide sequences encoding such enzymes. The sequences find use in preparing plants that show increased tolerance to the herbicide glyphosate. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided.
The Class III enzymes are characterized by having at least one domain selected from the domains listed below, herein referred to as Class III domains:
Domain I:
L-A-K-G-X1-S-X2-L-X3-G-A-L-K-S-D-D-T (SEQ ID NO:13), where X1 denotes lysine or threonine, where X2 denotes arginine or histidine, where X3 denotes serine or threonine;
Domain Ia:
L-A-K-G-X1, (SEQ ID NO:14), where X1 denotes lysine or threonine;
Domain Ib:
S-X1-L-X2 (SEQ ID NO:15), where X1 denotes arginine or histidine, where X2 denotes serine or threonine;
Domain Ic:











G-A-L-K-S-D-D-T;
(SEQ ID NO: 16)







Domain II:

E-P-D-X1-X2-T-F-X3-V-X4-X5-X6-G (SEQ ID NO:17), where X1 denotes aspartic acid or alanine, where X2 denotes serine or threonine, where X3 denotes valine or isoleucine, where X4 denotes threonine or glutamic acid or lysine, where X5 denotes serine or glycine, where X6 denotes glutamine or serine or glutamic acid or threonine;

Domain IIa:

E-P-D-X1-X2-T-F-X3-V (SEQ ID NO:18), where X1 denotes aspartic acid or alanine, where X2 denotes serine or threonine, where X3 denotes valine or isoleucine;

Domain IIb:

X1-X2-X3-G (SEQ ID NO:19), where X1 denotes threonine or glutamic acid or lysine, where X2 denotes serine or glycine, where X3 denotes glutamine or serine or glutamic acid or threonine;

Domain III:












RFLTAA;
(SEQ ID NO: 20)







Domain IV:

KRPI(G/M)P (SEQ ID NO:21)

K-R-P-I-X1-P, where X1 denotes glycine or methionine or leucine;

Domain V:

X1-G-C-P-P-V (SEQ ID NO:22), where X1 denotes threonine or serine;

Domain VI:

I-G-A-X1-G-Y-X2-D-L-T (SEQ ID NO:23), where X1 denotes arginine or lysine or leucine, and where X2 denotes isoleucine or valine;

Domain VII:

W-X1-V-X2-X3-T-G (SEQ ID NO:24), where X1 denotes arginine or lysine, where X2 denotes alanine or histidine or glutamic acid or serine, where X3 denotes proline or alanine;

Domain VIII:

E-P-D-A-S-A-A-T-Y-L-W-X1-A-X2-X3-L (SEQ ID NO:25), where X1 denotes alanine or glycine, where X2 denotes glutamic acid or glutamine, where X3 denotes valine or leucine or alanine;

Domain VIIIa:












E-P-D-A-S-A-A-T-Y-L-W;
(SEQ ID NO: 26)







Domain IX:

I-D-X1-G (SEQ ID NO:27), where X1 denotes isoleucine or leucine;

Domain X:

F-X1-Q-P-D-A-K-A (SEQ ID NO:28), where X1 denotes threonine or serine;

Domain XI:

X1-F-P-X2-X3-X4-A-X5-X6-X7-G-S-Q-M-Q-D-A-I-P-T-X8-A-V-X9A-A-F-N (SEQ ID NO:29), where X1 denotes glutamine or lysine or serine, where X2 denotes asparagine or histidine, where X3 denotes methionine or leucine, where X4 denotes proline or glutamine, where X5 denotes threonine or glutamic acid or valine, where X6 denotes valine or isoleucine, where X7 denotes aspartic acid or valine, where X8 denotes leucine or isoleucine, where X9 denotes leucine or isoleucine;

Domain XIa:

X1-F-P-X2-X3-X4-A (SEQ ID NO:30), where X1 denotes glutamine or lysine or serine, where X2 denotes asparagine or histidine, where X3 denotes methionine or leucine, where X4 denotes proline or glutamine;

Domain XIb:

X1-X2-X3-G-S-Q-M-Q-D-A-I-P-T-X4-A-V-X5A-A-F-N (SEQ ID NO:31), where X1 denotes threonine or glutamic acid or valine, where X2 denotes valine or isoleucine, where X3 denotes aspartic acid or valine, where X4 denotes leucine or isoleucine, where X5 denotes leucine or isoleucine;

Domain XIc:












G-S-Q-M-Q-D-A-I-P-T;
(SEQ ID NO: 32)







Domain XII:

P-V-R-F-X1-X2-X3-X4-N-L-R-V-K-E-C-D-R-X5 (SEQ ID NO:33), where X1 denotes valine or threonine, where X2 denotes glutamic acid or glycine, where X3 denotes leucine or isoleucine, where X4 denotes alanine or glutamic acid, where X5 denotes isoleucine or valine;

Domain XIIa:












P-V-R-F;
(SEQ ID NO: 34)







Domain XIIb:

X1-X2-X3-X4-N-L-R-V-K-E-C-D-R-X5 (SEQ ID NO:35), where X1 denotes valine or threonine, where X2 denotes glutamic acid or glycine, where X3 denotes leucine or isoleucine, where X4 denotes alanine or glutamic acid, where X5 denotes isoleucine or valine;

Domain XIIc:












N-L-R-V-K-E-C-D-R;
(SEQ ID NO: 36)







Domain XIII:

E-G-D-D-L-X1-X2 (SEQ ID NO:37), where X1 denotes leucine or isoleucine, where X2 denotes valine or isoleucine;

Domain XIV:

X1-P-X2-L-A-G (SEQ ID NO:38), where X1 denotes aspartic acid or asparagine, where X2 denotes alanine or serine or threonine;

Domain XV:

A-X1-I-D-X2-X3-X4-D-H-R (SEQ ID NO:39), where X1 denotes leucine or serine or glutamic acid, where X2 denotes threonine or serine, where X3 denotes histidine or phenylalanine, where X4 denotes alanine or serine;

Domain XVI:

F-A-L-A-X1-L-K-X2-X3-G-I (SEQ ID NO:40), where X1 denotes glycine or alanine, where X2 denotes isoleucine or valine, where X3 denotes serine or glycine or alanine or lysine;

Domain XVIa:

F-A-L-A-X1-L-K (SEQ ID NO:41), where X1 denotes glycine or alanine;

Domain XVIb:

L-K-X1-X2-G-I (SEQ ID NO:42), where X1 denotes isoleucine or valine, where X2 denotes serine or glycine or alanine or lysine; and

Domain XVII:

-X1-P-X2-C-V-X3-K (SEQ ID NO:43), where X1 denotes asparagine or aspartic acid, where X2 denotes alanine or aspartic acid, where X3 denotes alanine or glycine.

Domain XVIII:

X1-S-L-G-V (SEQ ID NO:44), where X1 denotes alanine or serine or proline.

The above domains set forth in SEQ ID NOS:13-44 were identified by aligning Class III sequences that share at least 50% sequence identity. In some embodiments at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 of these sequence domains are present.
Using the methods of the invention and the identified domains, additional proteins (for example, SEQ ID NOS:2, 4, 46 and 48) which confer glyphosate tolerance can be identified. These proteins include known proteins as well as newly identified proteins (for example, SEQ ID NOS:10, 12, 56, and 59).
By “glyphosate” is intended any herbicidal form of N-phosphonomethylglycine (including any salt thereof) and other forms that result in the production of the glyphosate anion in planta. An “herbicide resistance protein,” “herbicide tolerant protein,” or a protein resulting from expression of an “herbicide resistance-” or “herbicide tolerance-” encoding polynucleotide includes proteins that confer upon a cell the ability to tolerate a higher concentration of an herbicide than cells that do not express the protein, or to tolerate a certain concentration of an herbicide for a longer period of time than cells that do not express the protein. A “glyphosate resistance protein” or a “glyphosate tolerant protein” includes a protein that confers upon 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 time than cells that do not express the protein. By “tolerate” or “tolerance” is intended either to survive, or to carry out essential cellular functions such as protein synthesis and respiration in a manner that is not readily discemable from untreated cells.
Isolated Polynucleotides, and Variants and Fragments Thereof
One aspect of the invention pertains to isolated polynucleotides other than the polynucleotide sequences listed in SEQ ID NOS:1, 3, 7, 45, 47, and 53 encoding EPSP synthase enzymes having at least one Class III sequence domain of the invention. By “other than” is intended that the invention does not include the polynucleotide sequences set forth in the recited SEQ ID NOS.
The isolated polynucleotides of the present invention comprise nucleotide sequences encoding herbicide resistance proteins and polypeptides or biologically active portions thereof, as well as polynucleotides sufficient for use as hybridization probes to identify herbicide resistance-encoding polynucleotides. As used herein, the term “polynucleotide” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The polynucleotide can be single-stranded or double-stranded DNA.
Nucleotide sequences of the invention include those characterized by the domains included above. The information used in identifying these domains include sequence alignments of glyphosate-sensitive EPSPS molecules. The sequence alignments were used to identify regions of homology between the sequences and to identify the Class III domains that are characteristic of Class III EPSPS enzymes.
Variants of the domains are also encompassed within the scope of the invention (for example SEQ ID NO:55). Such variants include sequences sharing at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity and are contained in a DNA molecule that imparts glyphosate tolerance.
An “isolated” or “purified” polynucleotide or protein, or 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 other chemicals when chemically synthesized. Preferably, an “isolated” polynucleotide is free of sequences (for example, protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For purposes of the invention, “isolated” when used to refer to polynucleotides excludes isolated chromosomes. For example, in various embodiments, the isolated glyphosate resistance-encoding polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flanks the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. An herbicide resistance protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-herbicide resistance protein (also referred to herein as a “contaminating protein”).
Polynucleotides that are fragments of these herbicide resistance-encoding nucleotide sequences are also encompassed by the present invention (for example, SEQ ID NOS:57, 58, and 60). By “fragment” is intended a portion of the nucleotide sequence encoding an herbicide resistance protein (for example, SEQ ID NO:59). A fragment of a nucleotide sequence may encode a biologically active portion of an herbicide resistance protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. Polynucleotides that are fragments of an herbicide resistance nucleotide sequence comprise at least about 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 up to the number of nucleotides present in a full-length herbicide resistance-encoding nucleotide sequence disclosed herein. By “contiguous” nucleotides is intended nucleotide residues that are immediately adjacent to one another.
Fragments of the nucleotide sequences of the present invention generally will comprise at least one of the Class III domains described herein, and will encode protein fragments that retain the biological activity of the full-length glyphosate resistance protein; i.e., herbicide-resistance activity. By “retains herbicide resistance activity” is intended that the fragment will have at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the herbicide resistance activity of the full-length glyphosate resistance protein disclosed herein as SEQ ID NO:6. Methods for measuring herbicide resistance activity are well known in the art. See, for example, U.S. Pat. Nos. 4,535,060, and 5,188,642, each of which are herein incorporated by reference in their entirety.
A fragment of an herbicide resistance-encoding nucleotide sequence that encodes a biologically active portion of a protein of the invention will encode at least about 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 herbicide resistance protein of the invention. Importantly, the fragment will comprise at least one of the Class III domains described herein.
Herbicide resistance proteins of the present invention are those characterized as Class III or fragments or variants thereof that retain activity. The term “sufficiently identical” is intended an amino acid or nucleotide sequence that has at least about 60% or 65% sequence identity, at least about 70% or 75% sequence identity, at least about 80% or 85% sequence identity, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
To determine the percent identity of two amino acid sequences or of two polynucleotides, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions)×100). In one embodiment, the two sequences are the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to GDC-like polynucleotides of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to herbicide resistance protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-2402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. See www.ncbi.nlm.nih.gov. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, Calif.). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non-limiting example of a software program useful for analysis of ClustalW alignments is GENEDOCTM. GENEDOCTM (Karl Nicholas) allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package (available from Accelrys, Inc., San Diego, Calif.). When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
Unless otherwise stated, GAP Version 10, which uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used to determine sequence identity or similarity using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity or % similarity for an amino acid sequence using GAP weight of 8 and length weight of 2, and the BLOSUM62 scoring program. Equivalent programs may also be used. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
The invention also encompasses variant polynucleotides. “Variants” of the herbicide resistance-encoding nucleotide sequences include those sequences that encode the herbicide resistance protein disclosed herein but that differ conservatively because of the degeneracy of the genetic code, as well as those that are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the herbicide resistance proteins disclosed in the present invention as discussed below. Variant proteins encompassed by the present invention are biologically active, that is they retain the desired biological activity of the native protein, that is, herbicide resistance activity. By “retains herbicide resistance activity” is intended that the variant will have at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the herbicide resistance activity of the native protein. Methods for measuring herbicide resistance activity are well known in the art. See, for example, U.S. Pat. Nos. 4,535,060, and 5,188,642, each of which are herein incorporated by reference in their entirety.
The skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded herbicide resistance proteins, without altering the biological activity of the proteins. Thus, variant isolated polynucleotides can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.
For example, conservative amino acid substitutions may be made at one or more predicted, nonessential amino acid residues. A “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of an herbicide resistance protein without altering the biological activity, whereas an “essential” amino acid residue is required for 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 having 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Amino acid substitutions may be made in nonconserved 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 protein activity. However, one of skill in the art would understand that functional variants may have minor conserved or nonconserved alterations in the conserved residues.
Alternatively, variant nucleotide sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for the ability to confer herbicide resistance activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.
Using methods such as PCR, hybridization, and the like corresponding herbicide resistance sequences can be identified by looking for the conserved domains of the invention. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, St Louis, Mo.).
In a hybridization method, all or part of the herbicide resistance nucleotide sequence or a domain can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra. The so-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P, or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme co-factor. Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known herbicide resistance-encoding nucleotide sequence disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in the nucleotide sequence or encoded amino acid sequence can additionally be used. The probe typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, at least about 25, or at least about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800 consecutive nucleotides of herbicide resistance-encoding nucleotide sequence of the invention or a fragment or variant thereof. Methods for the preparation of probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), both of which are herein incorporated by reference.
Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, or less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of polynucleotides is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biolog—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Isolated Proteins and Variants and Fragments Thereof
Herbicide resistance proteins are also encompassed within the present invention. By “herbicide resistance protein” is intended a Class III protein having at least one of the domains set forth above, including, for example, SEQ ID NOS:10, 12, 56, and 59. Fragments, biologically active portions, and variants thereof are also provided, and may be used to practice the methods of the present invention.
“Fragments” or “biologically active portions” include polypeptide fragments comprising a portion of an amino acid sequence encoding an herbicide resistance protein and that retains herbicide resistance activity. A biologically active portion of an herbicide resistance protein can 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 recombinant techniques and evaluated for herbicide resistance activity. Methods for measuring herbicide resistance activity are well known in the art. See, for example, U.S. Pat. Nos. 4,535,060, and 5,188,642, each of which are herein incorporated by reference in their entirety.
By “variants” is intended proteins or polypeptides having an amino acid sequence that is at least about 60%, 65%, about 70%, 75%, about 80%, 85%, or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a Class III enzyme. Variants also include polypeptides encoded by a polynucleotide that hybridizes to the polynucleotide encoding a Class III enzyme, or a complement thereof, under stringent conditions. Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, retaining herbicide resistance activity. Methods for measuring herbicide resistance activity are well known in the art. See, for example, U.S. Pat. Nos. 4,535,060, and 5,188,642, each of which are herein incorporated by reference in their entirety.
Bacterial genes quite often possess multiple methionine initiation codons in proximity to the start of the open reading frame. Often, translation initiation at one or more of these start codons will lead to generation of a functional protein. These start codons can include ATG codons. However, bacteria such as Bacillus sp. also recognize the codon GTG as a start codon, and proteins that initiate translation at GTG codons contain a methionine at the first amino acid. Furthermore, it is not often determined a priori which of these codons are used naturally in the bacterium. Thus, it is understood that use of one of the alternate methionine codons may lead to generation of variants that confer herbicide resistance (for example, SEQ ID NO:59 encoded by SEQ ID NO:58). These herbicide resistance proteins are encompassed in the present invention and may be used in the methods of the present invention.
Antibodies to the polypeptides of the present invention, or to variants or fragments thereof, are also encompassed. 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, N.Y.; U.S. Pat. No. 4,196,265).
Transformation of Bacterial or Plant Cells
Transformation of bacterial cells is accomplished by one of several techniques known in the art, not limited to electroporation, or chemical transformation (See, for example, Ausubel (ed.) (1994) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., Indianopolis, Ind.). Markers conferring resistance to toxic substances are useful in identifying transformed cells (having taken up and expressed the test DNA) from non-transformed cells (those not containing or not expressing the test DNA). In one aspect of the invention, genes are useful as a marker to assess transformation of bacterial or plant cells.
Transformation of plant cells can be accomplished in similar fashion. By “plant” is intended whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen). “Transgenic plants” or “transformed plants” or “stably transformed” plants or cells or tissues refer to plants that have incorporated or integrated exogenous polynucleotide sequences or DNA fragments into the plant cell. By “stable transformation” is intended that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof.
The genes of the invention may be modified to obtain or enhance expression in plant cells. The herbicide resistance sequences of the invention may be provided in expression cassettes for expression in the plant of interest. “Plant expression cassette” includes DNA constructs that are capable of resulting in the expression of a protein from an open reading frame in a plant cell. The cassette will include in the 5′-3′ direction of transcription, a transcriptional initiation region (i.e., promoter) operably-linked to a DNA sequence of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants. In some embodiments, the transcriptional initiation region will cause the production of an RNA sequence that allows for sufficient expression of the encoded EPSPS enzyme to enhance the glyphosate tolerance of a plant cell transformed with the polynucleotide. By “sufficient expression” is intended that the transcription initiation region will provide for the expression of an amount of the glyphosate-resistant polypeptide of the invention (e.g., those containing a Class III domain) that will confer upon a plant or cell the ability to tolerate a higher concentration of glyphosate than plants or cells that do not contain or express the protein, or to tolerate a certain concentration of glyphosate for a longer time than plants or cells that do not contain or express the protein.
The cassette may additionally contain at least one additional gene to be cotransformed into the organism, such as a selectable marker gene. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites for insertion of the herbicide resistance sequence to be under the transcriptional regulation of the regulatory regions.
The promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is “native” or “homologous” to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is “foreign” or “heterologous” to the DNA sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention. “Heterologous” generally refers to the polynucleotide sequences that are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like. By “operably linked” is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the polynucleotide sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
Often, such constructs will also contain 5′ and 3′ untranslated regions. Such constructs may contain a “signal sequence” or “leader sequence” to facilitate co-translational or post-translational transport of the peptide of interest to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus, or to be secreted. For example, the gene can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum. By “signal sequence” is intended a sequence that is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. By “leader sequence” is intended any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a sub-cellular organelle. Thus, this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. The plant expression cassette can also be engineered to contain an intron, such that mRNA processing of the intron is required for expression.
By “3′ untranslated region” is intended a nucleotide sequence located downstream of a coding sequence. Polyadenylation signal sequences (for example, polyadenylated nucleotides) and other sequences encoding regulatory signals capable of affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor are 3′ untranslated regions. By “5′ untranslated region” is intended a nucleotide sequence located upstream of a coding sequence.
Other upstream or downstream untranslated elements include enhancers. Enhancers are nucleotide sequences 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 enhancer region and the 35S enhancer element.
The termination region may be native with the transcriptional initiation region, may be native with the herbicide resistance sequence of the present invention, or may be derived from another source. Convenient 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.
Where appropriate, the gene(s) may be optimized for increased expression in the transformed host cell. That is, the genes can be synthesized using host cell-preferred codons for improved expression, or may be synthesized using codons at a host-preferred codon usage frequency. Generally, the GC content of the gene will be increased. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are known in the art for synthesizing host-preferred genes. See, for example, U.S. Pat. Nos. 6,320,100; 6,075,185; 5,380,831; and 5,436,391, U.S. Published Application Nos. 20040005600 and 20010003849, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
In one embodiment, the polynucleotides of interest are targeted to the chloroplast for expression. In this manner, 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 gene product 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. In some embodiments, the polynucleotides of the invention encode a fusion polypeptide comprising an amino-terminal chloroplast transit peptide and the EPSPS enzyme. A “fusion polypeptide” can be generated, for example, by removing the stop codon from the polynucleotide sequence encoding a first polypeptide, then appending the polynucleotide sequence encoding a second polypeptide in frame such that the resulting polynucleotide sequence will then be expressed by a cell as a single polypeptide.
The polynucleotides of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the polynucleotides of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.
Typically this “plant expression cassette” will be inserted into a “plant transformation vector.” By “transformation vector” is intended a DNA molecule that is necessary for efficient transformation of a cell. Such a molecule may consist of one or more expression cassettes, and may be organized into more than one “vector” DNA molecule. For example, binary vectors are plant transformation vectors that utilize two non-contiguous DNA vectors to encode all requisite cis- and trans-acting functions for transformation of plant cells (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.
This plant transformation vector may be comprised of one or more DNA vectors needed for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that are comprised of more than one contiguous DNA segment. These vectors are often referred to in the art as “binary vectors.” Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a “gene of interest” (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the gene of interest are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as in understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science, 5:446-451). Several types of Agrobacterium strains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.
Plant Transformation
Methods of the invention involve introducing a nucleotide construct into a plant. By “introducing” is intended to present to the plant the nucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not require that a particular method for introducing a nucleotide construct to a plant is used, only that the nucleotide construct gains access to the interior of at least one cell of the plant. 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 transferring heterologous DNA into target plant cells (e.g. immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene and in this case “glyphosate”) to recover the transformed plant cells from a group of untransformed cell mass. In such processes, glyphosate-resistant polypeptides comprising one or more Class III domains of the present invention may be used as selectable marker.
Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent (e.g. “glyphosate”). The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grow into mature plant and produce fertile seeds (e.g. 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 cultured routinely. A general description of the techniques and methods for generating transgenic plants are 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 contains many cells, both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non-transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants. Then molecular and biochemical methods will be used for confirming the presence of the integrated heterologous gene of interest in the genome of transgenic plant.
Generation of transgenic plants may be performed by one of several methods, including but not limited to introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, and various other non-particle direct-mediated methods (e.g. 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.
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 relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as “transgenic seed”) having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
Plants
The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum. Preferably, plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).
This invention is particularly suitable for any member of the monocot plant family including, but not limited to, maize, rice, barley, oats, wheat, sorghum, rye, sugarcane, pineapple, yams, onion, banana, coconut, and dates.
Evaluation of Plant Transformation
Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of the heterologous gene(s) in the plant genome is confirmed by various methods such as analysis of polynucleotides, proteins and metabolites associated with the integrated gene.
PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of the incorporated gene(s) at the earlier stage before transplanting into the soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.
Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated in an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or “blot” then is probed with, for example, radiolabeled 32p target DNA fragment to confirm the integration of introduced gene in the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).
In Northern analysis, RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, 2001, supra). Expression of RNA encoded by the sequence of the invention is then tested by hybridizing the filter to a radioactive probe derived from a GDC, by methods known in the art (Sambrook and Russell, 2001, supra)
Western blot and biochemical assays and the like may be carried out on the transgenic plants to determine the presence of protein encoded by the herbicide resistance gene by standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present on the herbicide resistance protein.
The present invention is also drawn to methods for identifying an EPSP synthase with glyphosate resistance activity. The methods involve determining whether certain conserved sequence domains are present in the amino acid sequence of an EPSP synthase. The presence of one or more of the domains listed above.
Predicting Protein Function From Sequence
Using the methods of the invention and the identified domains, additional polypeptides (for example, SEQ ID NOS:2, 4, 46 and 48) which confer glyphosate tolerance can be identified. These additional polypeptides can be identified by searching sequence databases containing EPSP synthase sequences, and/or by alignment of polypeptide sequences of EPSP synthase to search for the presence of Class III domains. These polypeptides include known polypeptides as well as newly identified polypeptides. It is understood that some modification of these domains is tolerated in nature without disrupting the glyphosate resistance conferring nature of these domains, and are therefore equivalent to the domains listed herein.
In general, there are four levels of protein structure: the primary structure, which consists of the linear chain of amino acids, or the polypeptide sequence; the secondary structure, which is given by the α-helices, β-strands, and turns that the protein folds into; the tertiary structure, which is made up of simple motifs that have combined to form compact globular domains; and the quaternary structure, which can comprise several amino acid chains or subunits. When predicting function from sequence, it is important to identify the functionally important motifs or patterns. Protein domains with similar folds often share the same molecular function (Hegyi and Gerstein (1999) J. Mol. Biol. 288:147-164; Moult and Melamud (2000) Curr. Opin. Struct. Biol. 10:384-389; Shakhnovich et al. (2003) J. Mol. Biol. 326:1-9). Identification of domains important to protein function can be done by multiple sequence alignment.
Three-dimensional structure can be predicted by homology modeling, i.e., by using a sequence homologue (>25% sequence identity) with an experimentally determined 3D structure. The three-dimensional structure of, for example, E. coli EPSP synthase (AroA) is well known (Shönbrunn et al. (2001) Proc. Natl. Acad. Sci. USA 98:1375-1380). This structure is based on the crystallization of AroA with glyphosate and shikimate 3-phosphate.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
Example 1
Isolation of EPSPS Genes
Genes coding for class III EPSPS enzymes have been isolated from seven different bacteria (Klebsiella pneumoniae, Agrobacterium radiobacter, Rhizobium sp., Brevundomonas vesicularis, Agrobacterium tumefaciens, Pseudomonas syringae, and Brucella/Ochrobactrum).
The DNA coding sequence and the amino acid sequence of the grg8 open reading frame are provided in U.S. Patent Application No. 60/640,195, filed Dec. 29, 2004, and provided in SEQ ID NO:7 and SEQ ID NO:8 of this application, respectively.
The DNA coding sequence and amino acid sequence of the grg12 open reading frame are provided in SEQ ID NOS:57 and 10 and SEQ ID NOS:58 and 59 of this application, respectively.
The DNA coding sequence and amino acid sequence of the grg15 open reading frame are provided in SEQ ID NO:11 and SEQ ID NO:12 of this application, respectively.
The DNA coding sequence and amino acid sequence of the grg6 open reading frame are provided in GenBank Accession Number AE016853, bases 1,140,091 through 1,141,347, and provided in SEQ ID NO:1 and SEQ ID NO:2 of this application, respectively.
The DNA coding sequence and amino acid sequence of the grg9 open reading frame are provided in GenBank Accession Number NC—003304 bases 628,398 through 629,675, and provided in SEQ ID NO:3 and SEQ ID NO:4 of this application, respectively.
The DNA coding sequence and amino acid sequence of the grg7 open reading frame are provided in SEQ ID NO:45, and SEQ ID NO:46 of this application, respectively.
The DNA coding sequence and amino acid sequence of the grg5 open reading frame are provided in GenBank Accession Number NC—005773, bases 1 through 1257, and provided in SEQ ID NO:47 and SEQ ID NO:48 of this application, respectively.
The DNA coding sequence and amino acid sequence of the maize EPSPS open reading frame are provided in GenBank Accession Number X63374 (gi:1524383), bases 1 through 1335, and the protein sequence is provided in SEQ ID NO:50 of this application.
The DNA coding sequence and amino acid sequence of a bacterial EPSPS disclosed in International Patent Application WO2005014820 are provided in SEQ ID NO:53 and SEQ ID NO:54 of this application, respectively.
The DNA coding sequence and amino acid sequence of the grg7m1 open reading frame are provided in SEQ ID NO:55 and SEQ ID NO:56 of this application, respectively.
Example 2
Cloning the EPSP Synthase Gene From Pseudomonas syringae pv Tomato Strain DC3000
The EPSP synthase coding sequence was PCR-amplified from genomic DNA of Pseudomonas syringae pv. tomato strain DC3000 (ATCC BAA-871) using the following primers: CAGAGATCTGGCATGCGACCTCAAGCCACTCTC (upper, SEQ ID NO:61) and CAGGGCGCGCCTCAGCGCTGAACACTCACCC (lower, SEQ ID NO:62). The resultant 1.3 kb PCR product was digested with Bgl II and Asc I, ligated into modified pUC18 which had been digested with BamH I and Asc I, then electroporated into DH5α cells. Plasmid DNA was prepared from ampicillin resistant colonies and analyzed by restriction digest. One clone was chosen for further analysis. The DNA sequence of the insert was determined using techniques well known in the art and found to be 100% identical to the published sequence for strain DC3000 (Genbank accession number AE016853 bases 1,140,091 through 1,141,347). This plasmid was named pAX703, and the EPSPS ORF was named grg6.
Plasmid pAX703 was transformed into ΔaroA E. coli cells and found to complement the deletion. This demonstrated that grg6 encodes a functional EPSP synthase.
Example 3
Cloning the EPSP Synthase Gene From Agrobacterium radiobacter Strain C58
The EPSP synthase coding sequence was PCR-amplified from genomic DNA of Agrobacterium tumefaciens strain C58 (ATCC 33970) using the following primers: CAGGGATCCGGCATGATCGAACTGACCATCACCC (upper, SEQ ID NO:63) and CAGGGCGCGCCTCAGTGCTGCGGCTCGGCAGCG (lower, SEQ ID NO:64). The resultant 1.3 kb PCR product was digested with BamH I and Asc I, ligated into modified pUC18 which had been digested with BamH I and Asc I, then electroporated into DH5α cells. Plasmid DNA was prepared from ampicillin resistant colonies and analyzed by restriction digest. One clone was chosen for further analysis. The DNA sequence of the insert was determined using techniques well known in the art and found to be 100% identical to the published sequence for strain C58 (Genbank accession number NC—003304 bases 628398 through 629675). This plasmid was named pAX702, and the C58 EPSPS ORF was named grg9.
Plasmid pAX702 was transformed into ΔaroA E. coli cells and found to complement the deletion. This demonstrated that grg9 encodes a functional EPSP synthase.
Example 4
Testing grg6 and grg9 for Resistance to Glyphosate
Plasmids pAX703 and pAX702, containing grg6 and grg9, respectively, were transformed into E. coli cells and streaked onto M63 agar medium containing various concentrations of glyphosate. The vector plasmid pUC18 was used as a glyphosate-sensitive control. The results are presented in Table 1 below and demonstrate that expression of grg6 or grg9, confers resistance to high levels of glyphosate.







TABLE 1







Growth of E. coli expressing grg6 or grg9 in the


presence of glyphosate.









Glyphosate Concentration













Plasmid
Gene
0 mM
50 mM
100 mM







pUC18
(none)
++
−
−



pAX703
grg6
++
++
++



pAX702
grg9
++
++
++










Example 5
Cloning the EPSP Synthase Gene from Pseudomonas syringae pv syrinaae Strain B728a
The EPSP synthase coding sequence was PCR-amplified from a single well isolated colony of Pseudomonas syringae pv syringae strain B728a using the following primers: CAGGGATCCGGCATGCGACCTCAAGCCACCCTC (upper, SEQ ID NO:65) and CAGGGCGCGCCTCAGCGCTGAACACTCACAC (lower, SEQ ID NO:66). The resultant 1.26 kb PCR product was digested with appropriate restriction enzymes and ligated into the vector pRSF1b, then electroporated into E. coli cells. Plasmid DNA was prepared from ampicillin resistant colonies and analyzed by restriction digest. One clone was chosen for further analysis and designated as pAX1923. The DNA sequence of the open reading frame in pAX1923 was determined and found to be identical to the published sequence of the EPSPS from Pseudomonas syringae pv. syringae strain B728a. Thus, we designated this open reading frame as grg7.
Similarly, a separate 1.26 kb PCR product was digested with BamH I and Asc I, ligated into modified pUC18 which had been digested with BamH I and Asc I, then electroporated into DH5α cells. Plasmid DNA was prepared from ampicillin resistant colonies and analyzed by restriction digest. One clone was chosen for further analysis. The DNA sequence of the insert of pAX712 was determined using techniques well known in the art and found to contain 3 nucleotide changes when compared to the published DNA sequence of Pseudomonas syringae pv syringae strain B728a (Genbank Accession Number NZ_AABP02000003, bases 39,901 through 41,400). These three DNA nucleotide changes result in a protein with one amino acid change relative to the hypothetical protein encoded by the published sequence. The open reading frame from strain B728a encoding an EPSPS as identified in plasmid pAX712 was designated grg7m1 (SEQ ID NO:55; grg7m1 protein sequence set forth in SEQ ID NO:56).
Example 6
Cloning the EPSP Synthase Gene from Pseudomonas syringae pv phaseolicola strain 1448a
Sequence data for the EPSPS of Pseudomonas syringae pv phaseolicola strain 1448a was obtained from The Institute for Genomic Research website at www.tigr.org. The EPSP synthase coding sequence of Pseudomonas syringae pv phaseolicola strain 1448a was PCR-amplified from genomic DNA of Pseudomonas syringae pv phaseolicola strain 1448a (ATCC BAA-978) using the following primers: CAGGGATCCGGCATGCGACCTCAAGCCACCCTC (upper, SEQ ID NO:67) and AGAGGCGCGCCTCAGCGCTGAACACGCACC (lower, SEQ ID NO:68), designed based on the DNA sequence available from The Institute for Genomic Research (“TIGR”, personal communication). The resultant 1.26 kb PCR product was digested with BamH I and Asc I, ligated into modified pUC 18 which had been digested with BamH I and Asc I, then electroporated into DH5α cells. Plasmid DNA was prepared from ampicillin resistant colonies and analyzed by restriction digest. One clone was chosen for further analysis, and designated pAX713. The DNA sequence of the insert of pAX713 was determined using techniques well known in the art and found to be 100% identical to the published DNA sequence of Pseudomnonas syringae pv. phaseolicola strain 1448a (performed by The Institute for Genomic Research (TIGR), and available online in electronic form at www.tigr.org). This plasmid was named pAX713, and the open reading frame from strain 1448a encoding an EPSPS as identified in plasmid pAX713 was designated grg5.
Example 7
Testing of grg5 and grg7 for Resistance to Glyphosate
Plasmids pAX713, pAX1923, and pAX712, containing grg5, grg7, and grg7m1, respectively, were transformed into E. coli cells and streaked onto M63 agar medium containing various concentrations of glyphosate. The vector plasmid pUC18 was used as a glyphosate-sensitive control. The results are presented in Table 2 below and demonstrate that expression of grg5, grg7, or grg7m1 confers resistance to high levels of glyphosate.







TABLE 2







Growth of E. coli expressing grg5, grg7, or grg7m1 in the


presence of glyphosate.









Glyphosate Concentration













Plasmid
Gene
0 mM
50 mM
100 mM







pUC18
(none)
++
−
−



pAX713
grg5
++
++
++



pAX1923
grg7
++
++
++



pAX712
grg7m1
++
++
++










Example 8
Isolation of ATX20019
ATX20019 was isolated by plating samples of soil on HEPES Mineral Salts Medium (HMSM) containing glyphosate as the sole source of phosphorus. Since HMSM contains no aromatic amino acids, a strain must be resistant to glyphosate in order to grow on this media.
Two grams of soil were suspended in approximately 10 ml of water, vortexed for 15 seconds and permitted to settle for 15 minutes. A 10 μl loopful of this suspension was added to 3 ml of HMSM supplemented with 10 mM glyphosate (pH 7.0). HMSM contains (per liter): 10 g glucose, 2 g NH4SO4, 9.53 g HEPES, 1.0 ml 0.8 M MgSO4, 1.0 ml 0.1 M CaCl2, 1.0 ml Trace Elements Solution (In 100 ml of 1000× solution: 0.1 g FeSO4.7H2O, 0.5 mg CuSO4.5H2O, 1.0 mg H3BO3, 1.0 mg MnSO4.5H2O, 7.0 mg ZnSO4.7H2O, 1.0 mg MoO3, 4.0 g KCl). The culture was grown in a shaker incubator for four days at 28° C. and then 20 μl was used to inoculate 2.5 ml of fresh HMSM containing 10 mM glyphosate as the only phosphorus source. After two days, 20 μl was used to inoculate another fresh 2.5 ml culture. After 5 days, 20 μl was used to inoculate a fresh 2.5 ml culture. After sufficient growth, the culture was plated onto solid media by streaking a 1 μl loop onto the surface of agar plate containing HMSM agar containing 10 mM glyphosate as the sole phosphorus source and stored at 28° C. The culture was then replated for isolation. One particular strain, designated ATX20019, was selected due to its ability to grow in the presence of high glyphosate concentrations. ATX 20019 was determined to be a member of Ochrobactrum sp./Brucella sp. by sequencing of the 16S rDNA and comparison against a database.
Example 9
Preparation and Screening of Cosmid Libraries
Total DNA was extracted from a culture of ATX20019 using methods commonly known in the art. The DNA was partially digested with restriction enzyme Sau3A1 and ligated with SuperCos (Stratagene) vector fragment according to the manufacturer's directions. Ligation products were packaged into phage particles using GigaPack III XL packaging extract (Stratagene), transfected into E. coli aroA-cells. E. coli aroA- is a strain in which the native aroA gene, encoding EPSP synthase, has been deleted. This strain cannot grow on M63 medium because it requires exogenously supplied aromatic amino acids. The presence of a cosmid which contains an EPSP synthase gene can genetically complement the aroA-phenotype, that is, allow the strain to grow on M63 medium without exogenously supplied aromatic amino acids.
The transfected cells were plated on M63 agar medium containing 50 μg/ml kanamycin M63 agar medium containing 100 mM KH2PO4, 15 mM (NH4)2SO4, 50 μM CaCl2, 1 μM FeSO4, 50 μM MgCl2, 55 mM glucose, 25 mg/liter L-proline, 10 mg/liter thiamine HCl, sufficient NaOH to adjust the pH to 7.0, and 15 g/liter agar. Two colonies which grew on this medium were identified. Cosmid DNA was prepared from each of these colonies and re-transformed into E. coli aroA-cells. In each case, cells retransformed with cosmid DNA grew on M63 medium in the presence of 0 or 10 mM glyphosate while cells containing empty SuperCos vector did not. This confirms that the cosmids are able to complement the aroA-phenotype and able to confer resistance to glyphosate. These cosmids were named pAX1100 and pAX1101. The cosmids appeared to be identical by restriction digest analysis using two different enzymes. One cosmid, pAX1101, was selected for further characterization.
Example 10
Identification of grg12 in Cosmid pAX1101
To identify the gene(s) responsible for the glyphosate-resistance shown by cosmid pAX1101, DNA from this clone was mutagenized with transposable elements. In this method, one identifies clones that have suffered transposon insertions, and have lost the ability to confer glyphosate resistance. The location of the transposon insertions identifies the open reading frame responsible for the glyphosate resistance phenotype.
Cosmid pAX1101 was subjected to in vitro transposon mutagenesis using an EZ::TN Insertion Kit (Epicentre, Madison, Wis.) and the manufacturer's protocol. This process randomly inserts a transposon fragment into the cosmid DNA and thus randomly disrupts the function of genes in the cosmid. The transposons contain a gene encoding resistance to an antibiotic, so transposon insertion clones may be selected by the ability to grow in the presence of that antibiotic. The locations of the transposon insertions may be determined by restriction fragment mapping or by sequencing with primers which anneal in the transposon.
Transposon insertion clones of pAX1101 were transformed into E. coli strain DH5α and plated on M63 medium containing glyphosate. Multiple clones were found which had lost the ability to grow in the presence of glyphosate, indicating that the transposon had disrupted the gene responsible for resistance.
The DNA sequence was determined for the region of pAX1101 containing the transposon insertions using sequencing methods well known in the art and is presented as SEQ ID NO:9. An open reading frame (ORF) was identified at bases 46 through 1380 of SEQ ID NO:9. This nucleotide sequence is provided as SEQ ID NO:57, and the corresponding amino acid sequence is provided as SEQ ID NO:58. Analysis of sequence data from eight transposon insertion picks that had lost resistance to glyphosate revealed that all were within the ORF. This indicates that the ORF encodes the resistance to glyphosate conferred by the cosmid. This gene was named grg12. Cosmid pAX1101 containing the grg12 ORF was deposited at the Agricultural Research Service Culture Collection (NRRL) Apr. 4, 2005, and assigned Accession No. B-30833.
Example 11
Homology of GRG12 With Other Proteins
GRG12 has homology to EPSP synthase enzymes. An alignment of the GRG12 amino acid sequence (SEQ ID NO:10) with the amino acid sequences of other EPSP synthases is shown in FIG. 1. Table 3 lists the percent identity of GRG12 to various EPSP synthases. Examination of the amino acid sequence (SEQ ID NO:10) revealed that it does not contain the four domains typical of Class II EPSP synthase enzymes. Thus it is a novel, non-Class II, glyphosate-resistant EPSP synthase.
GRG12 has highest amino acid homology to an EPSPS described in WO2005014820 (SEQ ID NO:54). GRG12 (SEQ ID NO:10) has 92% amino acid identity to the EPSPS described in WO2005014820.







TABLE 3







Amino Acid Identity of GRG12 to Other EPSP Synthases











Percent Identity to



Gene
GRG12







GRG8
61%



GRG7
65%



GRG6
65%



GRG9
60%



D. psychrophila
32%



K. pneumoniae
31%



E. coli
30%



H. influenzae
30%



A. fulgidus
22%



CP4
22%



A. pernix
21%



C. perfringens
21%



B. subtilis
17%



WO2005014820
92%










Further analysis of the grg12 DNA sequence (SEQ ID NO:9) revealed the presence of a second, shorter open reading frame (SEQ ID NO:58) beginning with a GTG start codon at nucleotide 142 of SEQ ID NO:9. Translation of this open reading frame results in a protein (SEQ ID NO:59) that is identical to residues 33-444 of SEQ ID NO:10, except that the start codon of SEQ ID NO:59 is a methionine instead of the valine present in SEQ ID NO:10. Alignment of SEQ ID NO:59 with known EPSPS proteins indicated that this protein contains all residues known to be critical to function as an EPSPS. Thus, this protein is likely to comprise a functional, glyphosate-resistant EPSPS enzyme, whereas a protein resulting from initiation of translation from a start codon internal to the highly conserved domains would be unlikely to be functional. SEQ ID NO:59 is 97% identical to the EPSPS described in WO2005014820 (SEQ ID NO:54).
The ability of both open reading frames (SEQ ID NOS:57 and 58) to encode functional EPSPS activity may be tested by amplifying each open reading frame by PCR, cloning the resulting PCR fragments into a plasmid vector under the control of a suitable promoter, inducing expression of protein from the open reading frame as known in the art, and comparing the ability of the expressed protein to complement the aroA-phenotype in E. coli and to confer resistance to glyphosate.
Example 12
Engineering of grg12 for Expression of GRG12 Protein in E. coli 
The grg12 open reading frame (ORF) was amplified by PCR, cloned into a slightly modified version of the plasmid vector pUC18 and transformed into E. coli strain DH5α. The modifications to pUC18 were as follows: a stop codon was inserted into the lacZ open reading frame and a ribosome binding site (to optimize translational initiation of the inserted grg12 ORF) was inserted upstream of the BamHI restriction site. Plasmid DNA was prepared and the presence and orientation of the grg12 insert was determined by restriction digest. One clone which contained the ORF in the forward orientation with respect to the lac promoter in the vector was named pAX1106 and was tested for the ability to confer resistance to glyphosate. E. coli cells harboring pAX1106 or empty vector plasmid were streaked onto M63 agar plates containing 0 to 200 mM glyphosate. The results are presented in Table 4 below. These results demonstrate that grg12 confers resistance to high levels of glyphosate.







TABLE 4







Growth of pAX1106 on M63 Glyphosate Agar









Glyphosate




Concentration


(mM)
pAX1106
Empty Vector












0
+
+


50
+
−


100
+
−


200
+
−









Example 13
Purification of GRG12 Expressed as a 6×His-Tagged Protein in E. coli 
The grg12 coding region is amplified by PCR using PFUULTRATM DNA polymerase (Stratagene). Oligonucleotides used to prime PCR are designed to introduce restriction enzyme recognition sites near the 5′ and 3′ ends of the resulting PCR product. The resulting PCR product is digested with appropriate restriction enzymes and the digested product is cloned into the 6×His-tag expression vector pRSF1b (Novagen). The resulting clone contains grg12 in the same translational reading frame as, and immediately C-terminal to, the 6×His tag. General strategies for generating such clones, and for expressing proteins containing 6×His-tag are well known in the art. The level of expression of GRG12 protein may be determined on an SDS-PAGE protein gel. GRG12 protein can be isolated by purification of the induced GRG12 protein by chromatography on, for example, Ni-NTA Superflow Resin (Qiagen), as per manufacturer's instructions.
Example 14
Isolation of ATX4150
ATX4150 was isolated by plating samples of soil on Enriched Minimal Media (EMM) containing glyphosate as the sole source of phosphorus. Since EMM contains no aromatic amino acids, a strain must be resistant to glyphosate in order to grow on this media.
Two grams of soil were suspended in approximately 30 ml of water, and sonicated for 30 seconds in an Aquasonic sonicator water bath. The sample was vortexed for 5 seconds and permitted to settle for 60 seconds. This process was repeated 3 times. 100 μl of this suspension was added to 3 ml of EMM supplemented with 4 mM glyphosate (pH 6.0). EMM contains (per 900 mls): 10 g sucrose, 2 g NaNO3, 1.0 ml 0.8 M MgSO4, 1.0 ml 0.1 M CaCl2, 1.0 ml Trace Elements Solution (In 100 ml of 1000× solution: 0.1 g FeSO4.7H2O, 0.5 mg CuSO4.5H2O, 1.0 mg H3BO3, 1.0 mg MnSO4.5H2O, 7.0 mg ZnSO4.7H2O, 1.0 mg MoO3, 4.0 g KCl). The culture was shaken on a tissue culture roller drum for sixteen days at 21° C. and then 100 μl was used to inoculate 3 ml of fresh EMM containing 4 mM glyphosate as the only phosphorus source. After five days, 100 μl was used to inoculate another fresh 3 ml culture. After a few days, the culture was plated onto solid media by streaking a 1 μl loop onto the surface of agar plate containing EMM agar containing 5 mM glyphosate as the sole phosphorus source. After a few days, colonies were replated for isolation onto EMM containing 5 mM glyphosate as the sole phosphorus source. One particular strain, designated ATX4150, was selected due to its ability to grow in the presence of high glyphosate concentrations.
Example 15
Preparation and Screening of Cosmid Libraries
Total DNA was extracted from a culture of ATX4150 using methods commonly known in the art. The DNA was partially digested with restriction enzyme Sau3A1 and ligated with SuperCos (Stratagene) vector fragment according to the manufacturer's directions. Ligation products were packaged into phage particles using GigaPack III XL packaging extract (Stratagene), transfected into E. coli aroA-cells E. coli aroA-, is a strain in which the native aroA gene, encoding EPSP synthase, has been deleted. This strain cannot grow on M63 medium because it requires exogenously supplied aromatic amino acids. The presence of a cosmid which contains an EPSP synthase gene can genetically complement the aroA-phenotype, that is, it allow the strain to grow on M63 medium without exogenously supplied aromatic amino acids.
The transfected cells were plated on M63 agar medium containing 50 μg/ml kanamycin M63 agar medium contains 100 mM KH2PO4, 15 mM (NH4)2SO4, 50 μM CaCl2, 1 μM FeSO4, 50 μM MgCl2, 55 mM glucose, 25 mg/liter L-proline, 10 mg/liter thiamine HCl, sufficient NaOH to adjust the pH to 7.0, and 15 g/liter agar. Five colonies which grew on this medium were identified. Cosmid DNA was prepared from each of these colonies and re-transformed into E. coli aroA-cells. In each case cells retransformed with cosmid DNA grew on M63 medium in the presence of 0 or 10 mM glyphosate while cells containing empty SuperCos vector did not. This confirms that the cosmids are able to complement the aroA-phenotype and able to confer resistance to glyphosate. One cosmid was selected for further characterization and was named pAX305.
Example 16
Identification of grg15 in Cosmid pAX305
To identify the gene(s) responsible for the glyphosate-resistance shown by cosmid pAX305, DNA from this clone was mutagenized with transposable elements. Cosmid pAX305 was subjected to in vitro transposon mutagenesis using an EZ::TN Insertion Kit (Epicentre, Madison, Wis.) and the manufacturer's protocol. Transposon insertion clones of pAX305 were transformed into E. coli and plated on M63 medium containing glyphosate. Multiple clones were found which had lost the ability to grow in the presence of glyphosate, indicating that the transposon had disrupted the gene responsible for resistance.
The DNA sequence was determined for the region of pAX305 containing the transposon insertions using sequencing methods well known in the art and is presented in SEQ ID NO:61. An open reading frame (ORF, nucleotide bases 77 through 1354 of SEQ ID NO:61) was identified. Analysis of sequence data from eight transposon insertion picks that had lost resistance to glyphosate revealed that all were within the ORF. This indicates that the ORF encodes the resistance to glyphosate conferred by the cosmid. This gene was named grg15. Cosmid pAX305 containing the grg15 ORF (SEQ ID NO:11) was deposited at the Agricultural Research Service Culture Collection (NRRL) on Apr. 20, 2005 and assigned Accession No. NRRL B-30838.
Example 17
Homology of GRG15 With Other Proteins
GRG15 has homology to EPSP synthase enzymes. An alignment of the GRG15 amino acid sequence (SEQ ID NO:12) with the amino acid sequences of other EPSP synthases is shown in FIG. 1. Table 5 lists the percent identity of GRG15 to various EPSP synthases. Examination of the amino acid sequence (SEQ ID NO:12) revealed that it does not contain the four domains typical of Class II EPSP synthase enzymes. Thus it is a novel, non-Class II, glyphosate-resistant EPSP synthase.







TABLE 5







Amino Acid Identity of GRG15 to Other EPSP Synthases











Percent Identity to



Gene
GRG15







GRG9
94%



GRG8
70%



GRG7
66%



GRG6
66%



GRG12
60%



K. pneumoniae
32%



E. coli
32%



H. influenzae
32%



D. psychrophila
31%



A. fulgidus
26%



A. pernix
26%



C. perfringens
24%



B. subtilis
24%



GRG1
23%



Agrobacterium Sp.
22%



CP4










Example 18
Homology Blocks Among Glyphosate Resistant Non-Class II Enzymes (Class III)
Comparison of the amino acid sequences of the GRG proteins (non-class II glyphosate resistant proteins) show that the GRG proteins have significant homology to one another, and are distinct from previously identified glyphosate resistant EPSP synthases (see FIG. 1).
Example 19
Identification of Conserved Domains
The amino acid sequences of seven known EPSP synthases were analyzed for conserved domains that were not present in either class I or class II EPSP synthases. The following domains are found in these EPSP synthases: Bold type denotes conserved residues only among this class, and italics denote residues conserved in all EPSPS enzymes.
Domain I:
L-A-K-G-X1-S-X2-L-X3-G-A-L-K-S-D-D-T (SEQ ID NO:13), where X1 denotes lysine or threonine, where X2 denotes arginine or histidine, where X3 denotes serine or threonine.
Domain Ia:
L-A-K-G-X1, (SEQ ID NO:14), where X1 denotes lysine or threonine.
Domain Ib:
S-X1-L-X2 (SEQ ID NO:15), where X1 denotes arginine or histidine, where X2 denotes serine or threonine.
Domain Ic:











G-A-L-K-S-D-D-T
(SEQ ID NO: 16)







Domain II:

E-P-D-X1-X2-T-F-X3-V-X4-X5-X6-G (SEQ ID NO: 17), where X1 denotes aspartic acid or alanine, where X2 denotes serine or threonine, where X3 denotes valine or isoleucine, where X4 denotes threonine or glutamic acid or lysine, where X5 denotes serine or glycine, where X6 denotes glutamine or serine or glutamic acid or threonine.

Domain IIa:

E-P-D-X1-X2-T-F-X3-V (SEQ ID NO:18), where X1 denotes aspartic acid or alanine, where X2 denotes serine or threonine, where X3 denotes valine or isoleucine.

Domain IIb:

X1-X2-X3-G (SEQ ID NO:19), where X1 denotes threonine or glutamic acid or lysine, where X2 denotes serine or glycine, where X3 denotes glutamine or serine or glutamic acid or threonine.

Domain III:












RFLTAA
(SEQ ID NO: 20)







Domain IV:

KRPI(G/M)P (SEQ ID NO:21)

K-R-P-I-X1-P, where X1 denotes glycine or methionine or leucine.

Domain V:

X1-G-C-P-P-V (SEQ ID NO:22), where X1 denotes threonine or serine.

Domain VI:

I-G-A-X1-G-Y-X2-D-L-T (SEQ ID NO:23), where X1 denotes arginine or lysine or leucine, and where X2 denotes isoleucine or valine.

Domain VII:

W-X1-V-X2-X3-T-G (SEQ ID NO:24), where X1 denotes arginine or lysine, where X2 denotes alanine or histidine or glutamic acid or serine, where X3 denotes proline or alanine.

Domain VIII:

E-P-D-A-S-A-A-T-Y-L-W-X1-A-X2-X3-L (SEQ ID NO:25), where X1 denotes alanine or glycine, where X2 denotes glutamic acid or glutamine, where X3 denotes valine or leucine or alanine.

Domain VIIIa:












E-P-D-A-S-A-A-T-Y-L-W
(SEQ ID NO: 26)







Domain IX:

I-D-X1-G (SEQ ID NO:27), where X1 denotes isoleucine or leucine.

Domain X:

F-X1-Q-P-D-A-K-A (SEQ ID NO:28), where X1 denotes threonine or serine,

Domain XI:

X1-F-P-X2-X3-X4-A-X5-X6-X7-G-S-Q-M-Q-D-A-I-P-T-X8-A-V-X9A-A-F-N (SEQ ID NO:29), where X1 denotes glutamine or lysine or serine, where X2 denotes asparagine or histidine, where X3 denotes methionine or leucine, where X4 denotes proline or glutamine, where X5 denotes threonine or glutamic acid or valine, where X6 denotes valine or isoleucine, where X7 denotes aspartic acid or valine, where X8 denotes leucine or isoleucine, where X9 denotes leucine or isoleucine.

Domain XIa:

X1-F-P-X2-X3-X4-A (SEQ ID NO:30), where X1 denotes glutamine or lysine or serine, where X2 denotes asparagine or histidine, where X3 denotes methionine or leucine, where X4 denotes proline or glutamine.

Domain XIb:

X1-X2-X3-G-S-Q-M-Q-D-A-I-P-T-X4-A-V-X5A-A-F-N (SEQ ID NO:31), where X1 denotes threonine or glutamic acid or valine, where X2 denotes valine or isoleucine, where X3 denotes aspartic acid or valine, where X4 denotes leucine or isoleucine, where X5 denotes leucine or isoleucine.

Domain XIc:












G-S-Q-M-Q-D-A-I-P-T
(SEQ ID NO: 32)







Domain XII:

P-V-R-F-X1-X2-X3-Y4-N-L-R-V-K-E-C-D-R-X5 (SEQ ID NO:33), where X1 denotes valine or threonine, where X2 denotes glutamic acid or glycine, where X3 denotes leucine or isoleucine, where X4 denotes alanine or glutamic acid, where X5 denotes isoleucine or valine.

Domain XIIa:












P-V-R-F
(SEQ ID NO: 34)







Domain XIIb:

X1-X2-X3-X4-N-L-R-V-K-E-C-D-R-X5 (SEQ ID NO:35), where X1 denotes valine or threonine, where X2 denotes glutamic acid or glycine, where X3 denotes leucine or isoleucine, where X4 denotes alanine or glutamic acid, where X5 denotes isoleucine or valine.

Domain XIIc:












N-L-R-V-K-E-C-D-R
(SEQ ID NO: 36)







Domain XIII:

E-G-D-D-L-X1-X2 (SEQ ID NO:37), where X1 denotes leucine or isoleucine, where X2 denotes valine or isoleucine.

Domain XIV:

X1-P-X2-L-A-G (SEQ ID NO:38), where X1 denotes aspartic acid or asparagine, where X2 denotes alanine or serine or threonine.

Domain XV:

A-X1-I-D-X2-X3-X4-D-H-R- (SEQ ID NO:39), where X1 denotes leucine or serine or glutamic acid, where X2 denotes threonine or serine, where X3 denotes histidine or phenylalanine, where X4 denotes alanine or serine.

Domain XVI:

F-A-L-A-X1-L-K-X2-X3-G-I (SEQ ID NO:40), where X1 denotes glycine or alanine, where X2 denotes isoleucine or valine, where X3 denotes serine or glycine or alanine or lysine.

Domain XVIa:

F-A-L-A-X1-L-K (SEQ ID NO:41), where X1 denotes glycine or alanine.

Domain XVIb:

L-K-X1-X2-G-I (SEQ ID NO:42), where X1 denotes isoleucine or valine, where X2 denotes serine or glycine or alanine or lysine.

Domain XVII:

-X1-P-X2-C-V-X3-K (SEQ ID NO:43), where X1 denotes asparagine or aspartic acid, where X2 denotes alanine or aspartic acid, where X3 denotes alanine or glycine.

Domain XVIII:

X1-S-L-G-V (SEQ ID NO:44), where X1 denotes alanine or serine or proline.

Example 20
Engineering grg12 for Plant Transformation
The grg12 open reading frame (ORF) is amplified by PCR from a full-length cDNA template. Hind III restriction sites are added to each end of the ORF during PCR. Additionally, the nucleotide sequence ACC is added immediately 5′ to the start codon of the gene to increase translational 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 PCR.
The plasmid containing the grg12 PCR product is digested with, for example, Hind III and the fragment containing the intact ORF is isolated. In this example, the fragment is cloned into the Hind III site of a plasmid, such as pAX200, which is a plant expression vector 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 from this intermediate plasmid is then subcloned into a plasmid such as pSB11 (Japan Tobacco, Inc.) to form a final plasmid, referred to herein as pSB11GRG12. pSB11GRG12 is organized such that the DNA fragment containing the promoter—grg12—terminator construct may be excised by appropriate restriction enzymes and also used for transformation into plants, for example, by aerosol beam injection. The structure of pSB11GRG12 is verified by restriction digests and gel electrophoresis and by sequencing across the various cloning junctions.
The plasmid is mobilized into Agrobacterium tumefaciens strain LBA4404 which also harbors the plasmid pSB1 (Japan Tobacco, Inc.), using triparental mating procedures well known in the art, and plating on media containing antibiotic. Plasmid pSB11GRG12 carries spectinomycin resistance but is a narrow host range plasmid and cannot replicate in Agrobacterium. Antibiotic resistant colonies arise when pSB11GRG12 integrates into the broad host range plasmid pSB1 through homologous recombination. The resulting cointegrate product is verified by Southern hybridization. The Agrobacterium strain harboring the cointegrate can be used to transform maize, for example, by the PureIntro method (Japan Tobacco).
Example 21
Transformation of grg12 Into Plant Cells
Maize ears are best collected 8-12 days after pollination. Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in size are preferred for use in transformation. Embryos are plated scutellum side-up on a suitable incubation media, such as DN62A5S media (3.98 g/L N6 Salts; 1 mL/L (of 1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casamino acids; 50 g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D). However, media and salts other than DN62A5S are suitable and are known in the art. Embryos are incubated overnight at 25° C. in the dark. However, it is not necessary per se to incubate the embryos overnight.
The resulting explants are transferred to mesh squares (30-40 per plate), transferred onto osmotic media for about 30-45 minutes, then transferred to a beaming plate (see, for example, PCT Publication No. WO/0138514 and U.S. Pat. No. 5,240,842).
DNA constructs designed to express GRG12 in plant cells are accelerated into plant tissue using an aerosol beam accelerator, using conditions essentially as described in PCT Publication No. WO/0138514. After beaming, embryos are incubated for about 30 min on osmotic media, and placed onto incubation media overnight at 25° C. in the dark. To avoid unduly damaging beamed explants, they are incubated for at least 24 hours prior to transfer to recovery media. Embryos are then spread onto recovery period media, for about 5 days, 25° C. in the dark, then transferred to a selection media. Explants are incubated in selection media for up to eight weeks, depending on the nature and characteristics of the particular selection utilized. After the selection period, the resulting callus is transferred to embryo maturation media, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light, and the process of regeneration is initiated by methods known in the art. The resulting shoots are allowed to root on rooting media, and the resulting plants are transferred to nursery pots and propagated as transgenic plants.
Materials







TABLE 6







DN62A5S Media









Components
Per Liter
Source





Chu's N6 Basal Salt
3.98 g/L
Phytotechnology Labs


Mixture (Prod. No. C 416)


Chu's N6 Vitamin Solution
1 mL/L
Phytotechnology Labs


(Prod. No. C 149)
(of 1000x Stock)


L-Asparagine
800 mg/L
Phytotechnology Labs


Myo-inositol
100 mg/L
Sigma


L-Proline
1.4 g/L
Phytotechnology Labs


Casamino acids
100 mg/L
Fisher Scientific


Sucrose
50 g/L
Phytotechnology Labs


2,4-D (Prod. No. D-7299)
1 mL/L
Sigma



(of 1 mg/mL Stock)









The pH of the solution is adjusted to pH 5.8 with 1N KOH/1N KCl, Gelrite (Sigma) is added at a concentration up to 3 g/L, and the media is autoclaved. After cooling to 50° C., 2 ml/L of a 5 mg/ml stock solution of silver nitrate (Phytotechnology Labs) is added.
Example 22
Transformation of grg12 Into Maize Plant Cells by Agrobacterium-Mediated Transformation
Ears are best collected 8-12 days after pollination. Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in size are preferred for use in transformation. Embryos are plated scutellum side-up on a suitable incubation media, and incubated overnight at 25° C. in the dark. However, it is not necessary per se to incubate the embryos overnight. Embryos are contacted with an Agrobacterium strain containing the appropriate vectors for Ti plasmid mediated transfer for about 5-10 min, and then plated onto co-cultivation media for about 3 days (25° C. in the dark). After co-cultivation, explants are transferred to recovery period media for about five days (at 25° C. in the dark). Explants are incubated in selection media for up to eight weeks, depending on the nature and characteristics of the particular selection utilized. After the selection period, the resulting callus is transferred to embryo maturation media, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light, and the process of regeneration is initiated as known in the art. The resulting shoots are allowed to root on rooting media, and the resulting plants are transferred to nursery pots and propagated as transgenic plants.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

## Claims

1. An isolated polynucleotide encoding a glyphosate tolerant EPSPS enzyme having at least 95% sequence identity to SEQ ID NO:10, 12, or 59.

2. The polynucleotide of claim 1, wherein said polynucleotide is selected from the group consisting of:
a) the nucleotide sequence of SEQ ID NO:9, 11, 56, 57, 58, or 60;
b) the herbicide resistance nucleotide sequence of the DNA insert of the plasmid deposited as Accession Nos. B-30833 or B-30838, or a complement thereof and
c) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:10, 12, 56, or 59.

3. The polynucleotide of claim 1, wherein said polynucleotide is linked to a promoter which functions in plant cells to cause the production of an RNA sequence that allows for sufficient expression of the encoded EPSPS enzyme to enhance the glyphosate tolerance of a plant cell transformed with the polynucleotide.

4. The polynucleotide of claim 3 which further comprises a 3′ non-translated region which functions in plant cells to cause the addition of a stretch of polyadenylated nucleotides to the 3′ end of the RNA sequence.

5. The polynucleotide of claim 3, wherein said promoter is heterologous with respect to the polynucleotide.

6. A polynucleotide of claim 1 in which the polynucleotide encodes a fission polypeptide comprising an amino-terminal chloroplast transit peptide and the EPSPS enzyme.

7. A method of producing genetically transformed plants which are tolerant toward glyphosate herbicide, comprising the steps of:
a) inserting into the genome of a plant cell a polynucleotide comprising:
i) a promoter which functions in plant cells to cause the production of an RNA sequence,
ii) a polynucleotide encoding a glyphosate tolerant EPSPS enzyme having at least 95% sequence identity to SEQ ID NO:10, 12, or 59;
iii) a 3′ non-translated polynucleotide which functions in plant cells to cause the addition of a stretch of polyadenylated nucleotides to the 3′ end of the RNA sequence;

b) obtaining a transformed plant cell; and
c) regenerating from the transformed plant cell a genetically transformed plant which has increased tolerance to glyphosate herbicide.

8. The method of claim 7, wherein obtaining the transformed plant comprises screening for an enhanced tolerance to glyphosate herbicide.

9. A method of claim 7 in which the polynucleotide encodes a fusion polypeptide comprising an amino-terminal chloroplast transit peptide and the EPSPS enzyme.

10. A glyphosate-tolerant plant cell comprising the polynucleotide of claim 5.

11. A glyphosate-tolerant plant cell of claim 10 selected from the group consisting of corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grape and turf grasses.

12. A glyphosate-tolerant plant comprising plant cells of claim 10.

13. A transgenic seed comprising the polynucleotide of claim 1.

14. The glyphosate-tolerant plant of claim 12 selected from the group consisting of corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grape and turf grasses.

15. A method of producing a genetically transformed plant which is tolerant toward glyphosate herbicide, comprising the steps of:
a) inserting into the genome of a plant cell a polynucleotide encoding a glyphosate tolerant EPSPS enzyme having at least 95% sequence identity to SEQ ID NO:10, 12, or 59;
b) obtaining transformed plant cells; and
c) regenerating from the transformed plant cell a genetically transformed plant which has increased tolerance toward glyphosate herbicide.