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WO2005003362A2 - Methodes destinees a conferer une resistance aux herbicides - Google Patents

Methodes destinees a conferer une resistance aux herbicides Download PDF

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Publication number
WO2005003362A2
WO2005003362A2 PCT/US2004/007169 US2004007169W WO2005003362A2 WO 2005003362 A2 WO2005003362 A2 WO 2005003362A2 US 2004007169 W US2004007169 W US 2004007169W WO 2005003362 A2 WO2005003362 A2 WO 2005003362A2
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Prior art keywords
cell
plant
decarboxylase
herbicide
nucleotide sequence
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WO2005003362A3 (fr
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Philip E. Hammer
Todd K. Hinson
Nicholas B. Duck
Michael G. Koziel
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Athenix Corp
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Athenix Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8275—Glyphosate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • N-phosphonomethylglycine commonly referred to as glyphosate
  • Glyphosate inhibits the enzyme that converts phosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid to 5-enolpyruvyl-3- phosphoshikimic acid.
  • PEP phosphoenolpyruvic acid
  • ESP synthase 5-enolpyruvylshikimate-3- phosphate synthase
  • Glyphosate inhibits many bacterial EPSP synthases, and thus is toxic to these bacteria.
  • certain bacterial EPSP synthases may have a high tolerance to glyphosate.
  • Plant cells resistant to glyphosate toxicity can be produced by transforming plant cells to express glyphosate-resistant EPSP synthases.
  • a mutated EPSP synthase from Salmonella typhimurium strain CT7 confers glyphosate resistance in bacterial cells, and confers glyphosate resistance on plant cells (U.S. Patent Nos. 4,535,060, 4,769,061, and 5,094,945).
  • glyphosate- resistant bacterial EPSP synthases to confer glyphosate resistance upon plant cells.
  • An alternative method to generate target genes resistant to a toxin is to identify and develop enzymes that result in detoxification of the toxin to an inactive or less active form. This can be accomplished by identifying enzymes that encode resistance to the toxin in a toxin-sensitive test organism, such as a bacterium.
  • Castle et al. (WO 02/36782 A2) describe proteins (glyphosate N- acetyltransferases) that are described as modifying glyphosate by acetylation of a secondary amine to yield N-acetylglyphosate.
  • 168:702-707 describe degradation of glyphosate by C-P Lyase to yield glycine and inorganic phosphate. While several strategies are available for detoxification of toxins, such as the herbicide glyphosate, as described above, new activities capable of degrading glyphosate are useful. Novel genes and genes conferring glyphosate resistance by novel mechanisms of action would be of additional usefulness. Single genes conferring glyphosate resistance by formation of non-toxic products would be especially useful. Further, genes conferring resistance to other herbicides, such as the sulfonylureas or imidazolinones, are useful.
  • the sulfonylurea and imidazolinine herbicides are widely used in agriculture because of their efficacy at low use rates against a broad spectrum of weeds, lack of toxicity to mammals, and favorable environmental profile (Saari et al. (1994) p. 83-139 in: Herbicide Resistance in
  • Crop plants which are resistant to more than one class of herbicides provide growers with flexibility in weed control options and are useful in preventing/managing the emergence of resistant weed populations. Plants containing a single trait that conferred tolerance to more than one class of herbicide would be particularly desirable. Thus, genes encoding resistance to more than one class of herbicide are useful. Thus, methods that result in degradation of herbicides to non-toxic forms are desired. Further, methods that achieve sufficient degradation to allow cells to grow in otherwise toxic concentrations of herbicide (“herbicide resistance”) are desired. Methods that confer "herbicide resistance" through the expression of a single protein would be preferred, since expression of a single protein in a cell such as a plant cell is technically less complex than the expression of multiple proteins. Further, in some instances, methods for conferring herbicide resistance that are compatible with, and/or improve the efficacy of other methods of conferring herbicide resistance, are desirable.
  • Decarboxylase enzymes that could be useful in conferring herbicide resistance include, but are not limited to, a pyruvate decarboxylase, a benzoylformate decarboxylase, an oxalyl-CoA decarboxylase, a 2-oxoglutarate decarboxylase, an indolepyruvate decarboxylase, a 5-guanidino-2-oxopentanoate decarboxylase, a phenylglyoxylate dehydrogenase (acylating), a pyruvate dehydrogenase (cytochrome), a pyruvate oxidase, a pyruvate dehydrogenase (lipoamide), an oxoglutarate dehydrogenase (lipoamide), a transketolase, a formaldehyde transketolase, an acetoin- ribose-5-phosphate transaldolase, a tartron
  • FIG. 2A shows growth of GDC-1 expressing cells at various concentrations of glyphosate as compared to vector and media only controls at 42 hours.
  • Figure 2B shows growth of GDC-2 expressing cells at various concentrations of glyphosate as compared to vector and media only controls at 42 hours. Growth was measured by absorbance at 600 nm.
  • Figure 3 A shows the HPLC column elution profile of C 14 from a sample not incubated with GDC-1.
  • Figure 3B shows the HPLC column elution profile of C 14 after incubation with 100 ng GDC-1.
  • compositions and methods for conferring resistance to an herbicide in a cell particularly in a plant cell or a bacterial cell.
  • the methods involve transforming the cell with a nucleotide sequence encoding an herbicide resistance gene, i particular, the methods of the invention are useful for preparing plant and bacterial cells that show increased tolerance to the herbicide glyphosate.
  • compositions include transformed plants, plant cells, plant tissues and seeds as well as transformed bacterial cells.
  • Glyphosate includes any herbicidal form of N-phosphonomethylglycine (including any salt thereof) and other forms that result in the production of the glyphosate anion in planta.
  • Glyphosate (or herbicide) resistance-conferring decarboxylase or “GDC” includes a DNA segment that encodes all or part of a glyphosate (or herbicide) resistance protein. This includes DNA segments that are capable of expressing a protein that confers glyphosate (herbicide) resistance to a cell.
  • An "herbicide resistance protein” or an “herbicide resistance protein molecule” or a protein resulting from expression of an "herbicide resistance-encoding nucleic acid molecule” 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 time than cells that do not express the protein.
  • a “glyphosate resistance 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.
  • 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 discernable from untreated cells.
  • decarboxylase is intended a protein, or gene encoding a protein, whose catalytic mechanism can include cleavage and release of a carboxylic acid.
  • Decarboxylase includes proteins that utilize thiamine pyrophoshate as a cofactor in enzymatic catalysis. Many such decarbolyases also utilize other cofactors, such as FAD.
  • TPP -binding domain is intended a region of conserved amino acids present in enzymes that are capable of utilizing TPP as a cofactor.
  • Plant tissue includes all known forms of plants, including undifferentiated tissue (e.g.
  • Plant expression cassette includes DNN constructs that are capable of resulting in the expression of a protein from an open reading frame in a plant cell. Typically these contain a promoter and a coding sequence. Often, such constructs will also contain a 3 ' untranslated region. Such constructs may contain a 'signal sequence' or 'leader sequence' to facilitate co-translational or post-translational transport of the peptide to certain intraceUular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus.
  • TPP cofactor thiamine pyrophosphate
  • a well-known example of a decarboxylation reaction involving TPP as a cofactor is the conversion of pyruvate to acetaldehyde and CO by the enzyme pyruvate decarboxylase.
  • N-terminal domain regions are often referred to as the N-terminal domain, central domain, and C-terminal domain, in reference to their position within the amino acid sequence (see for example, Hawkins et al. (1989) FEBS Letters 255:77-82; Arjunan et al (1996) J. Mol. Biol. 256:590-600; Bar-ilan et al. (2001) Biochemistry 40:11946-11954).
  • pyruvate decarboxylase pyruvate dehydrogenase, ⁇ :-ketoglutarate dehydrogenase, and acetolactate synthase each contain TPP-binding domains.
  • TPP-binding proteins can be identified by comparison of the amino acid sequence of a new protein with the amino acid sequence of known TPP- binding proteins. Aside from the presence of conserved domains, decarboxylase enzymes can also share significant amino acid homology in regions of their amino acid sequence other than the conserved domains. Thus, a high degree of amino acid conservation is suggestive of similar functional role.
  • Co-pending U.S. Application entitled “ GDC-1 Genes Conferring Herbicide Resistance”, filed concurrently herewith, and incorporated herein by reference describes the identification of a gene sequence referred to therein as GDC-1. The sequence of GDC-1 encodes an herbicide resistance protein, conferring resistance to the herbicide glyphosate.
  • GDC-2 Genes Conferring Herbicide Resistance
  • the sequence of GDC-2 encodes an herbicide resistance protein, conferring resistance to the herbicide glyphosate.
  • GDC-1 and GDC-2 contain TPP- binding domains. While not being bound by any particular mechanism of action, the homology of the protein sequences of GDC-1 and GDC-2 herbicide tolerance- conferring genes to TPP-binding decarboxylases, as well as biochemical data provided herein, suggests that GDC-1 and/or GDC-2 encode herbicide tolerance by reactions involving the cofactor TPP.
  • herbicide resistance proteins for use in the methods of the present invention are decarboxylase enzymes.
  • decarboxylase enzymes examples include the GDC-1 coding sequence, as disclosed in co-pending U.S. Application entitled “GDC-1 Genes Conferring Herbicide Resistance”, filed concurrently herewith, is the herbicide resistance protein.
  • GDC-2 coding sequence as disclosed in co-pending U.S. Application entitled “GDC-2 Genes Conferring Herbicide Resistance", filed concurrently herewith, is the herbicide resistance protein.
  • Methods of the invention also encompass variant nucleic acid molecules that are sufficiently identical to the sequences provided for representative decarboxylase enzymes.
  • “Variants” of the herbicide resistance-encoding nucleotide sequences include those sequences that encode the decarboxylase proteins disclosed herein but that differ conservatively because of the degeneracy of the genetic code, as well as those that are sufficiently identical as described below.
  • 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 decarboxylase proteins disclosed in the present invention as discussed below.
  • Variant proteins for use in the methods of the present invention are biologically active, that is they retain the desired biological activity of the native protein, that is, herbicide resistance activity.
  • herbicide resistance activity By “retains herbicide resistance activity” is intended that the variant will have at least about 30%>, preferably at least about 50%, more preferably at least about 70%, even more preferably 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. Patent Nos. 4,535,060, and 5,188,642, each of which are herein incorporated by reference in their entirety.
  • the term "sufficiently identical" is intended an amino acid or nucleotide sequence that has at least about 60% or 65%o sequence identity, preferably about 70% or 75%o sequence identity, more preferably about 80% ⁇ or 85%> sequence identity, most preferably 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.
  • 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.
  • the sequences are aligned for optimal comparison purposes.
  • 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.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
  • PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra.
  • the default parameters of the respective programs e.g., BLASTX and BLASTN
  • 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.
  • 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., 9865 Scranton Rd., San Diego, California, USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • GAP version 10 which used the algorithm of Needleman and Wunsch (1970) supra.
  • GAP Version 10 may be used with 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 and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 Scoring Matrix. 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 or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
  • conservative amino acid substitutions may be made at one or more predicted, preferably 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.
  • 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
  • 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 ability to confer herbicide resistance activity to identify mutants that retain activity.
  • the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.
  • the methods of the invention also encompass nucleic acid molecules comprising nucleotide sequences encoding partial-length herbicide resistance proteins. Nucleic acid molecules that are fragments of the herbicide resistance- encoding nucleotide sequences are also encompassed by the present invention. By “fragment” is intended a portion of the nucleotide sequence encoding an herbicide resistance protein. 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.
  • Nucleic acid molecules 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, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600 nucleotides, or up to the number of nucleotides present in a full-length herbicide resistance- encoding nucleotide sequence (for example, 2210 nucleotides for SEQ TD NO:l) depending upon the intended use.
  • a full-length herbicide resistance- encoding nucleotide sequence for example, 2210 nucleotides for
  • Fragments of the nucleotide sequences will encode protein fragments that retain the biological activity of the native herbicide resistance protein.
  • “retains herbicide resistance activity” is intended that the fragment will have at least about 30%, preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 80% of the herbicide resistance activity of the native herbicide resistance protein.
  • Methods for measuring herbicide resistance activity are well known in the art. See, for example, U.S. Patent 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, 450, 500, or 550 contiguous amino acids, or up to the total number of amino acids present in a full- length herbicide resistance protein for use with methods of the invention (for example, 575 amino acids for SEQ ED NO: 3).
  • DNA sequence of an herbicide resistance gene maybe altered by various methods, and that these alterations may result in DNA sequences encoding proteins with amino acid sequences different that that encoded by an herbicide resistance gene.
  • This protein may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of the herbicide resistance protein can be prepared by mutations in the DNA. This may also be accomplished by one of several forms of mutagenesis and/or in directed evolution. En some aspects, the changes encoded in the amino acid sequence will not substantially affecting function of the protein. Such variants will possess the desired herbicide resistance activity.
  • an herbicide resistance gene to confer herbicide resistance may be improved by one use of such techniques upon the compositions of this invention.
  • protein sequences added can include entire protein-coding sequences, such as those used commonly in the art to generate protein fusions.
  • Such fusion proteins are often used to (1) increase expression of a protein of interest (2) introduce a binding domain, enzymatic activity, or epitope to facilitate either protein purification, protein detection, or other experimental uses known in the art (3) target secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of Gram-negative bacteria, or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in glycosylation of the protein.
  • Variant nucleotide and amino acid sequences of the present invention also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling.
  • one or more different herbicide resistance protein coding regions can be used to create a new herbicide resistance protein possessing the desired properties.
  • libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • sequence motifs encoding a domain of interest may be shuffled between the herbicide resistance gene of the invention and other known herbicide resistance genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased glyphosate resistance activity.
  • Strategies for such DNA shuffling are known in the art.
  • Herbicide resistance genes may be identified by isolating DNA or cDNA from an organism, preferably an organism that is capable of growing in herbicidal or antibiotic concenti-ations of an herbicide.
  • a library of clones (DNA or cDNA clones) can be transformed into a test organism, such as a bacterium.
  • E. coli may function as a test organism.
  • the individual clones can be then grown on media containing the herbicide or antibiotic, at a concentration at which the test organism does not grow, or grows noticeably slower or to a noticeably lower density than cells grown in media lacking the herbicide.
  • the clones conferring tolerance of the test cells to the herbicide can then be identified.
  • the DNA sequences of the positive clones are analyzed, and compared to databases of known proteins such as the Genbank 'nr' database.
  • those positive clones with homology to known decarboxylases, or minimally having amino acid homology to a TPP-binding domain can be identified.
  • sets of DNA sequences of genes or gene fragments maybe screened, such as the Genbank database, or the Genbank ⁇ ST database, and genes likely to encode decarboxylases or likely to have TPP-binding domains may be identified.
  • the genes could be cloned into a vector in such a way that the gene is expressed in a test cell, such as an E. coli cell.
  • the cells expressing the genes could be tested at various concentrations of an herbicide, and those conferring resistance to an herbicide, such as glyphosate, could be identified.
  • a known sequence of a TPP-binding protein may be used to generate DNA probes. Then these DNA probes can be utilized to screen a library (libraries) composed of cloned DNA, or cloned cDNA from one or more organisms by methods known in the art for identifying homologous gene sequences.
  • the homologous genes (if needed) can be engineered to be expressed in a test cell (such as an E.
  • proteins having TPP-binding characteristics may be purified, for example, by covalently attaching TPP to a solid matrix, such as a bead, and adsorbing crude or partially purified protein extracts to the bead, washing the bead, and eluting the TPP-binding protein, for example by varying salt, pH, or other conditions that cause the TPP molecule to no longer bind the TPP-binding domain.
  • the protein purified in this way can identify gene(s) likely to have herbicide resistance properties by obtaining a partial amino acid sequence of the protein, for example by performing amino-terminal amino acid sequencing.
  • the gene encoding this protein may be cloned by methods known in the art. Genes containing such TPP- binding domains can also be identified directly, for example by phage display or cell surface display technologies. Phage display methods are based on expressing recombinant proteins or peptides fused to a phage coat protein. Such phage are then used to perform binding assays, and phage containing inserts conferring binding ability (such as by expression of a TPP-binding domain) are retained, and can be propagated using traditional phage bacteriology techniques.
  • Bacterial display is a modification of phage display based on expressing recombinant proteins fused to sorting signals that direct their incorporation on the cell surface.
  • Methods for phage display and bacterial display are well known in the art. For example, see Benhar (2001) Biotechnol. Adv. 19:1-33, or Hartley (2002) J. Recept. Signal Transduct. Res. 22:373-92, and references within.
  • TPP-binding proteins are capable of conferring herbicide resistance
  • additional TPP-binding enzymes may be identified by virtue of their amino acid homology
  • additional herbicide-resistance encoding proteins may be identified by testing one or all of the subset of known TPP- binding proteins by one or all of the assays described, in order to assess the herbicide resistance-conferring ability of the protein.
  • the DNA sequence of any of the known classes of TPP-binding proteins may be used to identify novel related proteins, which are also likely to bind TPP as a consequence of their catalytic role.
  • TPP-binding proteins By this way, the herbicide resistance conferring ability of such genes may be assessed. Additionally, corresponding herbicide resistance sequences can be identified by using methods such as PCR, hybridization, and the like. See, for example, Sambrook j., and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Lnnis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY). In a hybridization method, all or part of the herbicide resistance nucleotide sequence can be used to screen cDNA or genomic libraries.
  • hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, 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, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 consecutive nucleotides of herbicide resistance encoding nucleotide disclosed herein or a fragment or variant thereof.
  • probes for hybridization are generally known in the art and is disclosed in Sambrook and Russell, 2001, herein incorporated by reference.
  • an entire herbicide resistance sequence disclosed herein, or one or more portions thereof may be used as a probe capable of specifically hybridizing to corresponding herbicide resistance sequences and messenger RNAs.
  • probes include sequences that are unique and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length.
  • Such probes may be used to amplify corresponding herbicide resistance sequences from a chosen organism by PCR.
  • a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
  • 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 moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX 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. IX SSC at 60 to 65°C.
  • T m is reduced by about 1°C for each 1% of mismatching; thus, T m , 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 T m can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
  • Transformation of 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.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994)). 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).
  • 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).
  • Transformation of plant cells can be accomplished in similar fashion.
  • the organization of such constructs is well known in the art.
  • the herbicide resistance sequences used in the methods of the invention may be provided in expression cassettes for expression in the plant of interest.
  • the cassette will include 5' and 3' regulatory sequences operably linked to a sequence of the invention.
  • 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.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • the cassette may additionally contain at least one additional gene to be cotransformed into the organism.
  • 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 expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the invention, and a transcriptional and translational tenmnation region (i.e., termination region) functional in plants.
  • the promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence of the invention.
  • 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.
  • the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the gene product of interest to the chloroplasts.
  • 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 at " . (1987) Plant Physiol 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.
  • Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-l,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb.
  • Rubisco chloroplast small subunit of ribulose-l,5-bisphosphate carboxylase
  • EPSPS 5-(enolpyruvyl)shikimate-3-phosphate synthase
  • the nucleic acids 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. Ln this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831, herein incorporated by reference. Typically this 'plant expression cassette' will be inserted into a 'plant transformation vector'.
  • 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.
  • Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-med ted 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.
  • the selectable marker gene and the gene of interest are located between the left and right borders.
  • 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, polyethelene glycol, etc.
  • Many types of vectors can be used to transform plant cells for achieving herbicide resistance.
  • 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 appropropriate 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.
  • 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 maximum threshold level of selecting agent e.g. "glyphosate”
  • 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 including aerosol beam transformation (U.S. Published Application No. 20010026941; U.S. Patent No. 4,945,050; International Publication No. WO 91/00915; U.S. Published Application No. 2002015066), and various other non-particle direct-mediated methods (e.g. Hiei et ⁇ l. (1994) The Plant Journal 6: 271-282; Ishida et al.
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci.
  • heterologous foreign DNA Following integration of heterologous foreign DNA into plant cells, one then applies a maximum threshold level of herbicide in the medium to kill the untransformed cells and separate and proliferate the putatively transformed cells that survive from this selection treatment by transferring regularly to a fresh medium. By continuous passage and challenge with herbicide, one identifies and proliferates the cells that are transformed with the plasmid vector. 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. 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.
  • 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.
  • Southern Analysis Plant transformation is confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001). Ln 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 32 P target DNA fragment to confirm the integration of introduced gene in the plant genome according to standard techniques (Sambrook and Russell, 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook, J., and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) Expression of RNA encoded by the herbicide resistance gene is then tested by hybridizing the filter to a radioactive probe derived from an herbicide resistance gene, by methods known in the art (Sambrook and Russell, 2001)
  • Western blot and Biochemical assays may be carried out on the transgenic plants to confirm the determine the presence of protein encoded by the herbicide resistance gene by standard procedures (Sambrook, J., and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) using antibodies that bind to one or more epitopes present on the herbicide resistance protein.
  • Herbicide Resistant Plants in another aspect of the invention, one may generate transgenic plants expressing an herbicide resistance gene that are more resistant to high concentrations of herbicide than non-transformed plants. Methods described above by way of example may be utilized to generate transgenic plants, but the manner in which the transgenic plant cells are generated is not critical to this invention. Methods known or described in the art such as Agrobacterium-medi&ted transformation, biolistic transformation, and non-particle-mediated methods may be used at the discretion of the experimenter. Plants expressing an herbicide resistance gene may be isolated by common methods described in the art, for example by transformation of callus, selection of transformed callus, and regeneration of fertile plants from such transgenic callus. In such process, an herbicide resistance gene may be used as selectable marker.
  • any gene as a selectable marker so long as its expression in plant cells confers ability to identify or select for transformed cells.
  • Genes known to function effectively as selectable markers in plant transformation are well known in the art. The following examples are offered by way of illustration and not by way of limitation.
  • GDC-1 and GDC-2 Confer Glyphosate Resistance Upon Cells Starter cultures of E. coli containing GDC-1 (full), GDC-2, or vector alone were grown overnight in LB media, diluted 1 : 1000 into 3 ml M9 minimal media containing 0, 2, 5, 10, 20 and 30 mM glyphosate and grown at 37°C. Each strain was grown in triplicate at each concentration. OD 6 oo was measured at 0, 7, 24, and 28 hours after inoculation. Table 2 shows the OD 6 oo obtained for each construct at 28 hours after inoculation.
  • GDC-1 and GDC-2 are Both TPP-binding Decarboxylases Searches of DNA and protein sequence databases, as well as sequence analysis of the GDC-1 and GDC-2 proteins show that they are homologous to pyruvate decarboxylase and acetolactate synthases. See, respectively, co-pending U.S. Application entitled “GDC-1 Genes Conferring Herbicide Resistance”, and co- pending U.S. Application entitled “GDC-2 Genes Conferring Herbicide Resistance”, both filed concurrently herewith. These searches reveal that both both GDC-1 and
  • GDC-2 contain amino acid regions which are conserved among TPP-binding proteins, including pyruvate decarboxylases and acetolactate synthases. An alignment of GDC- 1 and GDC-2 with other known TPP-binding proteins is shown in Figure 1.
  • Example 3 Engineering GDC-1 andGDC-2 for expression in E.coli E. coli strains expressing GDC-1 and GDC-2 were engineered into a customized expression vector, pAX481.
  • pAX481 contains the pBR322 origin of replication, a chloramphenicol acetyl transferase gene (for selection and maintenance of the plasmid), the lad gene, the Ptac promoter and the rrnB transcriptional terminator.
  • the GDC-1 and GDC-2 open reading frames were amplified by PCR, using a high fidelity DNA polymerase, as known in the art.
  • GDC-1 and GDC-2 Confer Resistance to High Levels of Glyphosate E. coli strains containing either GDC-1 (pAX472) or GDC-2 (pAX473) expression vectors, or vector controls (pAX481), were grown to saturation in M63 media, and diluted into a 48-well plate by adding 40 ⁇ l of cells to 1 ml cultures.
  • Spectramaxl90 Spectrophotometer (Molecular Devices, Inc.). The absorbance of the cultures at 0 hours was consistently below 0.04. The table below shows the absorbance at 600 nM obtained from the individual cultures after 42 hours of incubation.
  • GDC 1 and GDC-2 confer glyphosate resistance upon sensitive cells
  • GDC-1 and GDC-2 do not Complement an aroA Mutation in E.coli
  • the E. coli aroA gene codes for EPSP synthase, the target enzyme for glyphosate.
  • EPSP synthase catalyzes the sixth step in the biosynthesis of aromatic amino acids in microbes and plants.
  • aroA mutants that lack an EPSP synthase do not grow on minimal media that lacks aromatic amino acids (Pittard and Wallace (1966) J. Bacteriol. 91:1494-508), but can grow in rich media, such as LB.
  • genes encoding EPSPS activity can restore ability to grow on glyphosate upon aroA mutant E.coli strains.
  • a test for genetic complementation of an aroA mutant is a highly sensitive method to test if a gene is capable of functioning as an EPSPS in E. coli.
  • Such tests for gene function by genetic complementation are known in the art.
  • a deletion of the aroA gene was created in E. coli XL-1 MRF' (Stratagene) by PCR/recombination methods known in the art and outlined by Datsenko and Wanner, (2000) Proc. Natl. Acad. Sci. USA 97:6640-6645. This system is based on the Red system that allows for chromosomal disruptions of targeted sequences.
  • ⁇ aroA grows on LB media (which contains all amino acids) and grows on M63 media supplemented with phenylalanine, tryptophan, and tyrosine, but does not grow on M63 minimal media (which lacks aromatic amino acids).
  • Example 6 Purification of GDC-1 Expressed as a 6xHis-tagged Protein inE. coli
  • the GDC-1 coding region (1,728 nucleotides) was amplified by PCR using ProofStartTM DNA polymerase. Oligonucleotides used to prime PCR were designed to introduce restriction enzyme recognition sites near the 5' and 3' ends of the resulting PCR product.
  • the resulting PCR product was digested with BarnH I and Sal I. BamH I cleaved the PCR product at the 5 ' end, and Sal I cleaved the PCR product at the 3 ' end.
  • the digested product was cloned into the 6xHis-tag expression vector pQ ⁇ -30 (Qiagen), prepared by digestion with BamH I and Sal I.
  • the resulting clone, pAX623, contained GDC-1 in the same translational reading frame as, and immediately C- terminal to, the 6xHis tag of pQE-30.
  • General strategies for generating such clones, and for expressing proteins containing 6xHis-tag are well known in the art.
  • the ability of this clone to confer glyphosate resistance was confirmed by plating cells of pAX623 onto M63 media containing 5 mM glyphosate. pAX623 containing cells gave rise to colonies, where cells containing the vector alone gave no colonies.
  • GDC-1 protein from pAX623 -containing cells was isolated by expression of GDC-l-6xHis-tagged protein in E. coli, and the resulting protein purified using Ni- NTA Superflow Resin (Qiagen) as per manufacturer's instructions.
  • Example 7 Assay of GDC-1 Pyruvate Decarboxylase Activity lOOng of GDC-1 protein was tested for activity in a standard pyruvate decarboxylase assay (Gounaris et al. (1971) J. of Biol Chem. 246:1302-1309).
  • This assay is a coupled reaction where in the first step the pyruvate decarboxylase (PDC) converts pyruvate to acetaldehyde and CO .
  • the acetaldehyde produced in this reaction is a substrate for alcohol dehydrogenase, which converts acetaldehyde and ⁇ - NADH to ethanol and /3-NAD.
  • PDC activity is detected by virtue of utilization of /3-NADH as decrease in absorbance at 340 nM in a spectrophotometer.
  • GDC-1 as well as a control enzyme (pyruvate decarboxylase, Sigma) were tested in this assay.
  • GDC-1 showed activity as a pyruvate decarboxylase, and the reaction rate correlated with the concentration of pyruvate in the assay.
  • Example 8 Assay of GDC-1 Ability to Modify Glyphosate The ability of GDC-1 to modify glyphosate in vitro was tested by incubating GDC-1 with a mixture of radiolabeled and non-labeled glyphosate, and analyzing the reaction products by HPLC. 100 ng of GDC-1 purified protein was incubated with 20,000 cpm of C 14 labeled glyphosate (NaOOCCH 2 NH 14 CH 2 PO 3 H 2; Sigma catalog #G7014), mixed with unlabelled glyphosate to a final concentration of 2 mM in a reaction buffer of 200 mM Na-Citrate, pH 6.0, 1 mM TPP, 2 mM MgCl 2 .
  • Example 9 Purification of GDC-2 Expressed as a 6xHis-tagged Protein in E. coli
  • the GDC-2 coding region (2,088 nucleotides) was amplified by PCR using ProofStartTM DNA polymerase (Qiagen). Oligonucleotides used to prime PCR were designed to introduce restriction enzyme recognition sites near the 5 ' and 3 ' ends of the resulting PCR product. The resulting PCR product was digested with BamH. I and Hind III. BamH I cleaved the PCR product at the 5' end, and Sal I cleaved the PCR product at the 3 ' end.
  • GDC-2 protein from pAX624-containing cells was isolated by expression of GDC-2-6xHis-tagged protein in E. coli, and the resulting protein purified using Ni- NTA Superflow Resin (Qiagen) as per manufacturer's instructions.
  • Example 10 Assay of GDC-2 Acetolactate Synthase Activity
  • Acetolactate synthases are decarboxylating enzymes that condense two pyruvate molecules to form acetolactate with the release of a CO 2 moiety from one of the pyruvate substrates.
  • the product acetolactate is converted to acetoin by incubation with 1% H 2 SO 4 for 15 minutes at 60°C followed by neutralization with KOH. The acetoin is then detected as described by Westerfeld (Westerfeld (1945) J. Biol.
  • Example 11 Engineering GDC-1 for Plant Transformation
  • ORF open reading frame
  • Hindlll restriction sites were added to each end of the ORF during PCR.
  • nucleotide sequence ACC was added immediately 5' to the start codon of the gene to increase translational efficiency (Kozak (1987) Nucleic Acids Research 15:8125-8148; and Joshi (1987) Nucleic Acids Research 15:6643- 6653).
  • the PCR product was cloned and sequenced, using techniques well known in the art, to ensure that no mutations were introduced during PCR.
  • the plasmid containing the GDC-1 PCR product was partially digested with Hind III and the 1.7 kb Hind III fragment containing the intact ORF was isolated.
  • GDC-1 contains an internal Hind III site in addition to the sites added by PCR.
  • This fragment was cloned into the Hind III site of plasmid pAX200, a plant expression vector containing the rice actin promoter (McElroy et al. (1991) Molecular General Genetics 231:150-160) and the PinEI terminator (An et al. (1989) The Plant Cell 1:115-122) .
  • the promoter - gene - terminator fragment from this intermediate plasmid was subcloned into LKho I site of plasmid pSB 11 (Japan Tobacco, Inc.) to form the plasmid pAX810.
  • pAX810 is organized such that the 3.45 kb DNA fragment containing the promoter - GDC-1 - terminator construct may be excised from pAX810 by double digestion with Kpnl and Xbal for transformation into plants using aerosol beam injection.
  • the structure of pAX810 was verified by restriction digests and gel electrophoresis and by sequencing across the various cloning junctions.
  • Example 12 Engineering GDC-2 for Plant Transformation
  • ORF open reading frame
  • Hind III restriction sites were added to each end of the ORF during PCR. Additionally, the nucleotide sequence ACC was added immediately 5' to the start codon of the gene to increase translational efficiency (Kozak (1987) 15:8125-8148; Joshi (1987) Nucleic Acids Research 15:6643-6653).
  • the PCR product was cloned and sequenced, using techniques well known in the art, to ensure that no mutations were introduced during PCR.
  • the plasmid containing the GDC-2 PCR product was digested with Hind III and the fragment containing the intact ORF was isolated.
  • Plasmid pAX810 was mobilized into Agrobacterium tumifaciens strain LBA4404 which also harbored the plasmid pSBl (Japan Tobacco, Inc.), using triparental mating procedures well known in the art, and plating on media containing spectinomycin. Plasmid pAX811 carries spectinomycin resistance but is a narrow host range plasmid and cannot replicate in Agrobacterium. Spectinomycin resistant colonies arise when pAX811 integrates into the broad host range plasmid pSBl through homologous recombination.
  • Example 13 Transformation of GDC-1 and GDC-2 into Plant Cells
  • Maize ears are collected 8-12 days after pollination. Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in size are used for transformation. Embryos are plated scutellum side-up on a suitable incubation media, such as
  • DN62A5S media (3.98 g/LN6 Salts; 1 mL/L (of lOOOx Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casaminoacids; 50 g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D).
  • media and salts other than DN62A5S are suitable and are known in the art. Embryos are incubated overnight at 25 °C in the dark.
  • the resulting explants are transferred to mesh squares (30-40 per plate), transferred onto osmotic media for 30-45 minutes, then transferred to a beaming plate (see, for example, PCT Publication No. WO/0138514 and U.S. Patent No. 5,240,842).
  • DNA constructs designed to express GDC-1, GDC-2 or GDC-1 and GDC-2 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 30 min on osmotic media, and placed onto incubation media overnight at 25°C in the dark.
  • explants 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 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.
  • Example 14 Transformation of GDC-1 and GDC-2 into Plant Cells by Ears are collected 8-12 days after pollination. Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in size are used for 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 5-10 min, and then plated onto co-cultivation media for 3 days (25°C in the dark).
  • explants After co-cultivation, explants are transferred to recovery period media for 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.

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Abstract

L'invention concerne des compositions et des méthodes destinées à conférer une résistance aux herbicides à des cellules de plantes et des cellules bactériennes. Ces méthodes consistent à transformer les cellules avec des séquences nucléotidiques codant pour des gènes de résistance aux herbicides. Plus particulièrement, on obtient une résistance aux herbicides par expression de protéines présentant une homologie avec des enzymes décarboxylases. Lesdites compositions comprennent des plantes, des graines et des tissus de plantes transformés, ainsi que des cellules bactériennes transformées.
PCT/US2004/007169 2003-03-10 2004-03-10 Methodes destinees a conferer une resistance aux herbicides Ceased WO2005003362A2 (fr)

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