HUP0600691A2 - Synthetic herbicide resistance gene - Google Patents

Description

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Synthetic herbicide resistance gene

The invention relates to a synthetic herbicide resistance gene, the use of the gene according to the invention for producing herbicide-resistant transgenic plants, and the use of the gene as a selection marker.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide used to control broadleaf weeds. 2,4-D is degraded by Alcaligenes eutrophus and other microorganisms. The gene encoding the first enzyme in the A. eutrophus 2,4-D degradation pathway is tfdA. This gene encodes a dioxygenase that catalyzes the conversion of 2,4-D to 2,4-dichlorophenol (DCP). DCP is much less toxic to plants than 2,4-D, and transgenic tobacco, cotton, and hardwood trees carrying the tfdA gene have been reported to have increased resistance to 2,4-D [Streber et al.: Bio/Technology 7 811 (1989); Lyon et al.: Plant Molec. Biol. 13 533 (1989); Bayley et al.: Theor. Appl. Genet. 83 645 (1992); Llewellyn and Last: Herbicide-resistant Crops, Chapter 10, pp. 159-174. ed. Duke, CRC Press, (1996); Last and Llewellyn: Weed Science 47 401 (1999); U.S. Patents 6,153,401, 6,100,446 and 5,608,147; and PCT Publication Nos. WO 98/38294 and WO 95/18862], However, no transgenic plants have been found to be tolerant to the levels of 2,4-D that may occur under agricultural conditions, see, for example, Last and Llewellyn, Weed Science 47 401 (1999). These authors suggest that codon optimization of the tdfA gene may increase tolerance levels, see ibid, p. 404. The tdfA gene has also been used as a selection marker to identify transformed plants and plant cells, see, for example, U.S. Patent No. 5,608,147 and PCT Publication No. WO 95/18862.

The invention relates to a DNA molecule comprising a synthetic DNA sequence.

The synthetic DNA sequence encodes an enzyme that degrades 2,4-dichlorophenoxyacetic acid to dichlorophenol. The synthetic DNA sequence comprises a sequence of natural microbial origin that encodes an enzyme in which at least a plurality of codons of the natural microbial origin sequence have been replaced with codons more favorable to the plant.

The invention also provides a DNA construct comprising a synthetic DNA sequence as described herein. In this construct, the synthetic DNA sequence is operably linked to sequences that regulate plant gene expression.

-2The invention also provides a transgenic plant or plant part. The transgenic plant or plant part comprises the synthetic DNA sequence functionally linked to sequences that regulate plant gene expression.

The invention also provides a method for controlling weeds in a field containing transgenic plants according to the invention. The method comprises applying to the field a given amount of an auxin herbicide, which amount is effective for controlling weeds in the field. The transgenic plants are resistant to the auxin herbicide because they contain and express the synthetic DNA sequence. In fact, this is the first time that transgenic plants have been produced that are resistant to levels of auxin herbicide that are substantially higher than those commonly used in agriculture for weed control.

The invention also provides methods for selecting transformed plants and plant cells. In the method for selecting transformed plant cells, a population of plant cells is first established. At least some of the plant cells in the population are transformed with the DNA construct of the invention. The resulting population of plant cells is then grown in a culture medium containing an auxin herbicide, the concentration of the herbicide being selected such that the transformed plant cells are able to proliferate and the non-transformed plant cells are unable to proliferate.

In the process for selecting transformed plants, a population of plants is established that is presumed to contain a transgenic plant according to the invention. An auxin herbicide is then applied to the population of plants, the amount of herbicide being selected such that the transformed plants are able to grow and the growth of non-transformed plants is inhibited.

Figure 1 is a diagram of pProPCISV-SAD.

Figure 2 is a lg7 diagram of pPZP21 l-PNPT-31.

Figure 3 is a diagram of pPZP21 l-PNPT-512g7.

Figure 4 is a diagram of pPZP21 l-PNPT-311-SAD.

Figure 5 is a diagram of pPZP211-PNPT-512-SAD.

In these figures, the abbreviations have the following meanings: SAD = 2,4-D degradation synthetic gene adapted to dicots; CDS = coding sequence; AMV-leader = 5’ untranslated leader sequence from the 35S transcript of alfalfa mosaic virus; PCISV-promoter = peanut chlorotic streak virus promoter; T-left = T-DNA left flanking sequence from the nopaline Ti plasmid of Agrobacterium tumefaciens pTiT37; 35SPolyA = 3’ polyadenylation (polyA) termination signal sequence from cauliflower mosaic virus (CaMV)

from the 35S transcript; NPTII = neomycin phosphotransferase II; g7PolyA = 3’ polyA termination signal from gene-7 within the T-left flanking sequence of an A. tumefaciens octopine plasmid; MCS = multiple cloning site; T-right = T-DNA right flanking sequence from the A. tumefaciens pTiT37 Ti plasmid.

The invention relates to a synthetic DNA sequence. The term synthetic is used herein to mean that the DNA sequence does not occur naturally.

The synthetic DNA sequence of the invention encodes an enzyme that degrades 2,4-dichlorophenoxyacetic acid (2,4-D) to dichlorophenol (DCP). The synthetic DNA sequence comprises a naturally occurring microbial sequence encoding the enzyme and in the DNA sequence at least a plurality of codons in the naturally occurring microbial sequence have been replaced with codons more advantageous to the plant.

As used herein, the term naturally occurring microbial sequence refers to the coding sequence of a naturally occurring microbial gene encoding an enzyme capable of degrading 2,4-D to DCP. Thus, as used herein, the naturally occurring microbial sequence may be the coding sequence of a cDNA or genomic clone isolated from a microorganism, or may be a chemically synthesized DNA molecule having a coding sequence identical to the sequence of the above clone, or may be a combination of such sequences.

Multi-enzyme pathways for 2,4-D degradation have been demonstrated in several bacterial genera, see, for example, Lyon et al. Plant Molec. Biol. 13 533 (1989) and references therein. Strains of Alcaligenes eutrophus have been the most extensively studied of these bacteria. The first enzyme in the A. eutrophus degradation pathway converts 2,4-D to DCP. This enzyme, often referred to as a monooxygenase but now recognized as a dioxygenase [Fukumori et al. J. Bacteriol. 175 2083 (1993)], is encoded by the tfdA gene. Thus, the naturally occurring microbial sequence may be the coding sequence of a cDNA or genomic clone encoding the tfdA dioxygenase. Such clones and their isolation are described in the following publications: Bayley et al.: Theor. Appl. Genet. 83 645 (1992); Lyon et al.: Plant Molec. Biol. 13 533 (1989); Streber et al.: J. Bacteriology 169 2950 (1987); Perkins and Lurquin: J. Bacteriology 170 5669 (1988); and U.S. Patents 6,100,446 and 6,153,401. See also: Maniatis et al.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982) and Sambrook et al.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989).

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Many bacteria are known to be able to degrade 2,4-D, including strains of Acinetobacter, Achromobacter, Alcaligenes, Arthrobacter, Corynebacterium, Flavobacterium, Pseudomona, and strains of Actinomycetes such as Nocardia subspecies and Streptomyces viridochromogenes [see, for example, Llewellyn and Last, Herbicide-Resistant Crops, Chapter 10, ed. Duke, CRC Press Inc. (1996); Bayley et al., Theor. Appl. Genet. 83 645 (1992); Lyon et al., Plant Molec. Biol. 13 533 (1989); and Streber et al., J. Bacteriology 169 2950 (1987); Loos, Degradation Of Herbicides, pp. 1-49. eds.: Kearney and Kaufman, Marcel Dekker Inc., New York (1969) and references cited therein]; additional strains of bacteria capable of degrading 2,4-D can be isolated using methods well known in the art (such as isolation from soils where 2,4-D is used in the culture propagation technique, see, for example: Loos: Degradation Of Herbicides, pp. 1-49. eds.: Kearney and Kaufman, Marcel Dekker Inc., New York (1969)). Additional eDNA and genomic clones encoding the enzyme that converts 2,4-D to DCP can be obtained from these additional bacteria in a similar manner as for the tfdA clones. See, for example: Bayley et al.: Theor. Appl. Genet. 83 645 (1992); Lyon et al.: Plant Molec. Biol. 13,533 (1989); Streber et al., J. Bacteriology 169, 2950 (1987); Perkins and Lurquin, J. Bacteriology 170, 5669 (1988); and U.S. Patents 6,100,446 and 6,153,401. See also Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989). Additionally or alternatively, isolated clones, portions thereof, or sequences derived therefrom can be used as probes for the identification and isolation of additional clones. See, for example, Perkins and Lurquin, J. Bacteriology 170, 5669 (1988); Bayley et al., Theor. Appl. Genet. 83,645 (1992); and U.S. Patents 6,100,446 and 6,153,401. See also Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989). The naturally occurring microbial sequence may be the coding sequence of one of these cDNA or genomic clones.

Yeasts and fungi are also known to degrade 2,4-D [see, for example, Llewellyn and Last, Herbicide-Resistant Crops, Chapter 10, ed. Duke, CRC Press Inc. (1996); Han and New, Soil Biol. Biochem. 26 1689 (1994); Donnelly et al., Applied And Environmental Microbiology 59 2642 (1993); Loos, Degradation Of Herbicides, pp. 1-49, ed. Kearney and Kaufman, Marcel Dekker Inc., New York (1969); and references cited therein]; additional strains of yeast and fungi capable of degrading 2,4-D can be isolated using methods well known in the art (such as soil·· · ** · · ♦

-5-Isolation from plants where 2,4-D is used in the culture propagation technique, see, for example, Loos: Degradation Of Herbicides, pp. 1-49. eds. Kearney and Kaufman, Marcel Dekker Inc., New York (1969) and Han and New: Soil Biol. Biochem. 26 1689 (1994)). Additional eDNA and genomic clones encoding the enzyme that converts 2,4-D to DCP can be obtained from yeast and fungi using methods well known in the art (see the references cited above for discussion of clone generation from bacteria) and the naturally occurring microbial sequence can be the coding sequence for one of these cDNA or genomic clones.

Furthermore, as noted above, the naturally occurring microbial sequence may be chemically synthesized in whole or in part. To accomplish this, the cDNA or genomic clone obtained as discussed in the preceding sections is sequenced using methods well known in the art. See, for example, Maniatis et al.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982) and Sambrook et al.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989). A synthetic DNA sequence containing the coding sequence of the eDNA or genomic clone may be prepared by chemical synthesis in whole or in part, using methods well known in the art, see, for example, Maniatis et al.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982) and Sambrook et al.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989). For example, DNA sequences can be synthesized using phosphoamidite chemistry using an automated DNA synthesizer. Also, the sequence of the A. eutrophus JMP134 tfdA gene is available [see, for example, Streber et al., J. Bacteriology 169 2950 (1987); and U.S. Patent Nos. 6,100,446 and 6,153,401, and GenBank (accession number M16730)], and a synthetic DNA sequence containing the coding sequence of the A. eutrophus tfdA gene can be prepared by total or partial chemical synthesis.

A preferred naturally occurring microbial sequence may be a naturally occurring bacterial sequence. As used herein, a naturally occurring bacterial sequence is a naturally occurring bacterial gene encoding an enzyme that degrades 2,4-D to DCP. Thus, the naturally occurring bacterial sequence may be a coding sequence of a cDNA or genomic clone isolated from a bacterium, a chemically synthesized DNA molecule having the same coding sequence as such a clone, or a combination of such sequences. Most preferably, the naturally occurring bacterial sequence is a coding sequence of a cDNA or genomic clone isolated from an A. eutrophus strain, or a chemically synthesized DNA molecule having the same coding sequence as such a clone.

-6 coding sequence, as such a clone, or it may be a combination of such sequences.

As noted above, at least a plurality of codons in the naturally occurring microbial sequence are replaced with codons that are preferred by the plant (referred to herein as plant-preferred codons). As used herein, the terms plant-preferred codon or plant-preferred codon are intended to refer to a codon that is more frequently used by a plant to encode a particular amino acid than the microbial codon encoding the particular amino acid. Preferably, the term plant-preferred codon is intended to refer to a codon that is most frequently used by the plant to encode the particular amino acid. Plant codon usage may refer to plants in general, to a class of plants (such as dicotyledons), to a specific type of plant (such as cotton or soybean), etc. The codon usage or preferences of a plant or group of plants may be inferred using methods known in the art. See, for example, Maximizing Gene Expression, 225-85. pp., eds.: Reznikoff and Gold, (1986); Perlak et al.: Proc. Natl. Acad. Sci. USA 88 3324 (1991); and PCT Publication Nos. WO 97/31115, WO 97/11086, European Patent Nos. 646643, 553494 and U.S. Patent Nos. 5,689,052, 5,567,862, 5,567,600, 5,552,299 and 5,017,692. For example, the codons used by the plant or plants which encode the set of different amino acids in a group of proteins expressed by the plant or plants - preferably in the case of proteins which are highly expressed - are tabulated. This can be accomplished by manual methods or by using software designed for this purpose (see, for example, PCT application WO 97/11086).

The use of plant-preferred codons in the plant in which the synthetic DNA sequence is expressed increases expression compared to the naturally occurring microbial sequence. Published literature indicates that codon usage affects gene expression in plants at the level of mRNA stability and translational efficiency. See, for example, Perlak et al.: Proc. Natl. Acad. Sci. USA 88 3324 (1991); Adang et al.: Plant Molec. Biol. 21 1131 (1993); Sutton et al.: Transgenic Res. 1 228 (1992). It is not necessary to replace all of the codons of a naturally occurring microbial sequence with codons preferred by the plant in order to achieve increased expression. However, it is preferable to replace at least the codons least preferred by the plant with codons preferred by the plant. In the description, the term least preferred codon by the plant refers to that codon-L ·*’·Γ

-7 is understood to mean the least commonly used in a naturally occurring microbial sequence for encoding a given amino acid by the plant or plants in question. Preferably, more than about 50 percent, most preferably at least about 80 percent, of the microbial codons are replaced with codons preferred by the plant.

Plant-preferred codons can be introduced into the naturally occurring microbial sequence by methods well known in the art. For example, site-directed mutagenesis can be used [see Perlak et al.: Proc. Natl. Acad. Sci. USA 88 3324 (1991)]. See also Maniatis et al.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982) and Sambrook et al.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989). However, plant-preferred codons are preferably introduced into the naturally occurring microbial sequence by chemical synthesis by synthesizing the entire DNA sequence encoding the 2,4-D degrading enzyme. More specifically, chemical synthesis is of great advantage when a large number of microbial codons are to be replaced by plant-preferred codons. Chemical synthesis has several additional advantages. For example, the use of chemical synthesis allows for further modifications to the sequence of the DNA molecule or the protein it encodes, such as optimizing expression (i.e. removing mRNA secondary structures that inhibit transcription or translation, removing unwanted potential polyadenylation sequences, and altering the A+T and G+C content), adding unique restriction sites at appropriate points, deleting protease cleavage sites, etc.

A synthetic DNA sequence containing plant-preferred codons in which at least one of the microbial codons has been replaced encodes the same amino acid sequence as the naturally occurring microbial sequence, provided that these substitutions are the only differences between the synthetic DNA sequence and the naturally occurring microbial sequence. However, the synthetic DNA sequence may also contain additional changes compared to the naturally occurring microbial sequence. For example, the synthetic DNA sequence may encode an enzyme that degrades 2,4-D to DCP but which has an altered amino acid sequence relative to the enzyme encoded by the unmutated naturally occurring microbial sequence due to one or more substitutions, additions, or deletions in the naturally occurring microbial sequence. Methods for making such substitutions, additions and deletions are believed to be well known in the art and have been described above.

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Test methods for determining whether 2,4-D has been degraded to DCP are well known in the art. See, for example, Streber et al.: J. Bacteriol. 169 2950 (1987); Perkins et al.: J. Bacteriol. 170 5669 (1988); Streber et al.: Bio/Technology 7 811 (1989); Lyon et al.: Plant Molec. Biol. 13 533 (1989); Bayley et al.: Theor. Appl. Genet. 83 645 (1992); Fukumori et al.: J. Bacteriol. 175 2083 (1993); Lyon et al.: Transgenic Res 2 162 (1993); Llewellyn and Last: In: Herbicide-Resistant Crops, Chapter 10, pp. 159-174, eds.: Duke, CRC Press (1996); Last and Llewellyn: Weed Science 47 401 (1999). However, resistance to 2,4-D and other auxin herbicides can also be used to demonstrate this transformation. See below and the references cited above.

The invention also provides DNA constructs comprising the DNA sequence operably linked to plant gene expression regulatory sequences. As used herein, the term DNA construct is understood to mean engineered (not naturally occurring) DNA molecules that are useful for delivering DNA into host cells and includes chimeric genes, expression cassettes and vectors.

As used herein, the term "operably linked" refers to the linking of DNA sequences (including the order of the sequences, the orientation of the sequences, and the relative positions of the different sequences relative to each other) in such a way that the protein encoded by them is expressed. Methods for functionally linking expression control sequences to coding sequences are well known in the art. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989).

As used herein, expression control sequences are DNA sequences that play a role in the regulation of transcription or translation in any way. Suitable expression control sequences and methods for their preparation and use are well known in the art.

The expression control sequences must include a promoter. The promoter may be any DNA sequence that exhibits transcriptional activity in selected plant cells, plant parts, or plants. The promoter may be inducible or constitutive. It may be a naturally occurring promoter or may consist of parts of different naturally occurring promoters, or may be partially or

fully synthetic promoter. Studies on promoter structure, such as that of Harley and Reynolds [Harley and Reynolds: Nucleic Acids Res. 15 2343 (1987)], can provide guidance in the design of promoters. However, the location of the promoter relative to the transcription start site can also be optimized. See, for example, Roberts et al.: Proc. Natl Acad. Sci. USA 76 760 (1979). Many suitable promoters for use in plants are known in the art.

For example, suitable constitutive promoters for use in plants include: promoters from plant viruses, such as the peanut chlorotic streak caulimovirus (PC1SV) promoter [U.S. Patent No. 5,850,019]; the 35S promoter from cauliflower mosaic virus (CaMV) [Odell et al., Nature 313,810 (1985)]; promoters from the Chorella virus methyltransferase genes [U.S. Patent No. 5,563,328]; and the full-length transcript promoter from the field fenugreek mosaic virus (FMV) [U.S. Patent No. 5,378,619]; promoters from genes such as rice actin [McElroy et al., Plant Cell 2, 163 (1990)]; ubiquitin [Christensen et al., Plant Mol. Biol. 12,619 (1989) and Christensen et al.: Plant Mol. Biol. 18,675 (1992)]; pEMU [Last et al.: Theor. Appl. Genet. 81,581 (1991)]; MAS [Velten et al.: EMBO J. 3,2723 (1984)]; maize histone H3 [Lepetit et al.: Mol. Gen. Genet. 231,276 (1992) and Atanassova et al.: Plant Journal 2,291 (1992)]; Brassica napus ALS3 [PCT publication number WO 97/41228]; and promoters of various Agrobacterium genes [see, for example, 4,771,002; 5,102,796; 5,182,200; US Patent No. 5,428,147],

Suitable inducible promoters for use in plants include: the promoter from the ACE1 system, which responds to copper [Mett et al.: Proc. Natl. Acad. Sci. USA 90 4567 (1993)]; the promoter of the maize In2 gene, which responds to benzenesulfonamide herbicide safety agents [Hershey et al.: Mol. Gen. Genetics 227 229 (1991) and Gatz et al.: Mol. Gen. Genetics 243 32 (1994)]; and the Tet repressor promoter from Tn10 [Gatz et al.: Mol. Gen. Genet. 227 229 (1991)]. A particularly preferred inducible promoter for use in plants is one that responds to an inducing agent to which plants do not normally respond. Examples of such an inducible promoter include an inducible promoter derived from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone [Schena et al.: Proc. Natl. Acad. Sci. USA 88 10421 (1991)]; or the recent use of a chimeric transcriptional activator - XVE - in an estrogen receptor-based inducible plant expression system, which is activated by estradiol.

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- 10vál [Zuo et al.: The Plant Journal 24 265 (2000)]. Further inducible promoters for use in plants are described in EP patent application number 332104 and PCT publication numbers WO 93/21334 and WO 97/06269.

Finally, promoters assembled from parts of other promoters and promoters synthesized in whole or in part may also be used. See, for example, Ni et al., Plant J. 7661 (1995) and PCT Publication No. WO 95/14098, which describe such promoters for use in plants.

The promoter may comprise, or be modified to comprise, one or more enhancer elements. Preferably, the promoter comprises a plurality of enhancer elements. Promoters comprising enhancer elements provide for higher levels of transcription compared to promoters which do not comprise them. Suitable enhancer elements for use in plants include the PC1SV enhancer element [U.S. Patent No. 5,850,019]; the CaMV 35S enhancer element [U.S. Patents Nos. 5,106,739 and 5,164,316] and the FMV enhancer element [Maiti et al.: Transgenic Res. 6 143 (1997)]. See also PCT Publication No. WO 96/23898 and Enhancers and Eukaryotic Expression (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1983).

For efficient expression, the coding sequences are preferably also operably linked to a 3' untranslated sequence. The 3' untranslated sequence includes a transcription terminator sequence and a polyadenylation sequence. The 3' untranslated region can be derived from the flanking regions of genes derived from Agrobacterium, plant viruses, plants, or other eukaryotes.

Suitable non-translated 3' sequences for plant applications include the following: cauliflower mosaic virus 35S gene, the phaseolin seed storage protein gene, the pea ribulose-biphosphate carboxylase small subunit E9 gene, the soybean 7S storage protein genes, the octopine synthase gene, and the nopaline synthase gene.

A 5' untranslated sequence is also used. The 5' untranslated sequence is a portion of an mRNA that extends from the 5' CAP site to the translation initiation codon. This region of the mRNA is required for translation initiation in plants and plays a role in the regulation of gene expression. Suitable 5' untranslated regions for use in plants include the following: a region from alfalfa mosaic virus, the cucumber mosaic virus coat protein gene, and tobacco mosaic virus.

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- 11 As mentioned above, the DNA construct may be a vector. The vector may contain one or more replication systems that allow it to replicate in host cells. Self-replicating vectors include plasmids, cosmids, and viral vectors. Alternatively, the vector may be an integrating vector that allows the integration of the synthetic DNA sequence encoding the 2,4-D degrading enzyme into the chromosome of the host cell. The vector may optionally have unique restriction sites for insertion of the DNA sequences. If a vector does not have unique restriction sites, it may be modified by introducing or removing restriction sites to make it more suitable for further manipulation.

The DNA constructs of the invention can be used to transform any type of plant cell (see below). It is essential to use a genetic marker to select transformed plant cells (selection marker). Selection markers typically allow the transformed cells to be recovered by negative selection (i.e., inhibiting the growth of cells that do not contain the selection marker) or to screen for the product encoded by the selection marker.

The most commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptll) gene, which can be isolated from Tn5 and which, when placed under the control of plant expression control signals, confers resistance to kanamycin [Fraley et al.: Proc. Natl. Acad. Sci. USA 80 4803 (1983)].

Another commonly used selectable marker gene is the hygromycin phosphotransferase gene, which confers resistance to the antibiotic hygromycin [Vanden Elzen et al., Plant Mol. Biol. 5:299 (1985)].

Other bacterial selectable marker genes that confer antibiotic resistance include gentamicin acetyltransferase, streptomycin phosphotransferase, aminoglycoside-3'-adenyltransferase, and the bleomycin resistance determinant [Hayford et al.: Plant Physiol. 86 1216 (1988); Jones et al.: Mol. Gen. Genet. 210 86 (1987); Sváb et al.: Plant Mol. Biol. 14 197 (1990); Hille et al.: Plant Mol. Biol. 7 171 (1986)]. Additional selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate, or bromoxynil [Comai et al.: Nature 317 741 (1985); Stalker et al.: Science 242 419 (1988); Hinchee et al.: Bio/Technology 6 915 (1988); Stalker et al.: J. Biol. Chem. 263 6310 (1988); and Gordon-Kamm et al.: Plant Cell 2 603 (1990)].

Additional selectable marker genes of non-bacterial origin can also be used in plant transformations. These genes include, for example: mouse dihydrofolate-re·· · ·« ·· ··

- 12-ductase, plant 5-enol-pyruvoyl-shikimate-3-phosphate synthase and plant acetolacetate synthesis [Eichholtz et al.: Somatic Cell Mol. Genet. 13 67 (1987); Shah et al.: Science 233 478 (1986); Charest et al.: Plant Cell Rep. 8 643 (1990); EP patent registration number 154,204],

Genes commonly used to screen for potentially transformed cells include: b-glucuronidase (GUS), b-galactosidase, luciferase, and chloramphenicol acetyltransferase [Jefferson: Plant Mol. Biol. Rep. 5 387 (1987); Teeri et al.: EMBO J. 8 343 (1989); Koncz et al.: Proc. Natl. Acad. Sci. USA 84 131 (1987); De Block et al.: EMBO J. 3 1681 (1984)], green fluorescent protein (GFP) [Chalfie et al.: Science 263 802 (1994); Haseloff et al.: TIG 11 328 (1995) and PCT Publication No. WO 97/41228]. Another approach to identifying relatively rare transformation events could be to use a gene encoding a dominant constitutive regulator of the Zea mays anthocyanin pigmentation pathway [Ludwig et al.: Science 247 449 (1990)].

In another aspect of the invention, resistance to an auxin herbicide can be used as a selectable marker for plants and plant cells. As used herein, the term auxin herbicide refers to phenoxy auxins (phenoxy herbicides) which include 2,4-D, 4-chlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxybutyric acid, 4-(2-methyl-4-chlorophenoxy)butyric acid, 2-(4-chlorophenoxy)propionic acid, 2-(2,4-dichlorophenoxy)propionic acid, 2-(2,4-dichlorophenoxy)propionic acid, 2-(2,4,5-trichlorophenoxy)propionic acid, and salts (including amine salts) and esters of these acids. Auxin herbicides are commercially available [see Crop Protection Reference, 11th edition, Chemical & Pharmaceutical Press, Inc., New York, NY, (1995)]. Preferred auxin herbicides include 2,4-D and its salts (including amine salts) and esters. By the term resistance is meant the ability of transformed plant cells to grow (survive, proliferate and regenerate as plants) when placed in a culture medium containing an auxin herbicide, where the level of herbicide is chosen to prevent non-transformed cells from growing. The term resistance also means the ability of transformed plants to grow after application of an auxin herbicide at a level that inhibits the growth of non-transformed plants.

Methods for selecting transformed plant cells are well known in the art. Briefly, in a population of plant cells (e.g., an explant or embryo suspension culture), at least some of the plant cells are transformed with the synthetic DNA sequence of the invention.

- 13 with a DNA construct. The resulting plant cell population is placed on a medium containing an auxin herbicide, the concentration of the herbicide being selected such that the transformed plant cells grow while the non-transformed plant cells do not. Appropriate concentrations of the auxin herbicide can be determined by experimental methods as is known in the art. In particular, for 2,4-D, this amount should be selected such that it inhibits the formation of random shoots from non-transformed plant cells and allows the formation of random shoots from transformed plant cells, as is apparently the case with the naturally occurring bacterial tfdA gene. See U.S. Patent No. 5,608,147 and PCT Publication No. WO 95/18862. Generally, the required amount of 2,4-D is in the concentration range of about 0.001 mg/L to about 5 mg/L of the culture medium, preferably in the concentration range of about 0.01 mg/L to 0.2 mg/L.

Methods for selecting transformed plants are also known in the art. Briefly, an auxin herbicide is applied to a population of plants that may contain one or more transgenic plants carrying the DNA construct of the invention that provides for 2,4-D degradation. The amount of auxin herbicide is selected such that the transformed plants grow and the growth of non-transformed plants is inhibited. The level of inhibition must necessarily be sufficient to readily distinguish transformed and non-transformed plants (i.e., the inhibition must be statistically significant). These amounts can be determined experimentally by methods known in the art. See also: Crop Protection Reference, 11th ed., Chemical & Pharmaceutical Press, Inc., New York, NY, (1995).

Selection based on resistance to auxin herbicides can be used to produce plants that are tolerant to 2,4-D and other auxin herbicides, in which case the use of another selection marker is not necessary. The absence of another selection marker is advantageous because it minimizes the number of foreign genes expressed.

Selection based on resistance to auxin herbicides can also be used to produce transgenic plants that express other desired genes. Many such genes are known and include genes encoding commercially valuable proteins as well as genes that confer improved agricultural properties on plants [see, for example, PCT Publication No. WO 97/41228, which is hereby incorporated by reference in its entirety].

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- 14The DNA constructs of the invention can be used for the transformation of various plant cells. The synthetic DNA sequence encoding the 2,4-D degrading enzyme and the selection marker - if a separate selection marker is used - can be located on the same or different DNA constructs. Preferably, they are arranged as a transcription unit on a single DNA construct, so that all the coding sequences are expressed together. At the same time, the desired gene(s) and the synthetic DNA sequence encoding the 2,4-D degrading enzyme - when resistance to the auxin herbicide is used as a selection marker - can be located on the same or different DNA constructs. Such constructs are prepared as described above.

Suitable host cells include any plant cell (see below). Preferably, a plant cell is used that does not normally degrade auxin herbicides. However, the invention can also be used to increase the level of degradation of auxin herbicides in plants that normally degrade such herbicides.

Thus, the transgenic plants, plant parts and plant cells of the invention include plants, plant parts and plant cells that do not normally degrade auxin herbicides, but which have been transformed according to the invention in such a way that they are able to degrade these herbicides and include the descendants, plant parts and plant cells of these transformed plants. The transgenic plants, plant parts and plant cells of the invention also include plants, plant parts and plant cells that normally degrade auxin herbicides, but which have been transformed according to the invention in such a way that they are able to degrade these herbicides better or more efficiently and include the descendants, plant parts and plant cells of these transformed plants.

As used herein, the term plant refers to a unicellular organism or a multicellular differentiated organism capable of photosynthesis, including algae, angiosperms (monocots and dicots), gymnosperms, bryophytes, mosses, and mosses. As used herein, the term plant part refers to parts of multicellular differentiated plants and includes seeds, pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, explants, etc. As used herein, the term plant cell refers to the structural and physiological unit of multicellular plants. Thus, the term plant cell refers to any cell that is of plant origin or part of a plant or derived from a plant. Examples of cells within the scope of the invention include:

- Differentiated cells that are part of a living plant, differentiated cells in culture, undifferentiated cells in culture and cells of undifferentiated tissues such as callus or tumor cells may be used.

Methods for transforming plant cells are well known in the art. For example, numerous methods have been developed for transforming plants, including biological and physical transformation protocols. See, for example, Miki et al.: Procedures for Introducing Foreign DNA into Plants in: Methods in Plant Molecular Biology and Biotechnology, pp. 67-88, ed.: Glick and Thompson, CRC Press, Inc., Boca Raton, (1993). Vectors and in vitro culture methods are also available for transforming plant cells or tissues and regenerating plants. See, for example, Gruber et al.: Vectors for Plant Transformation in: Methods in Plant Molecular Biology and Biotechnology, pp. 89-119, ed.: Glick and Thompson, CRC Press, Inc., Boca Raton, (1993).

The most widely used mechanism for introducing expression vectors into plants is based on the natural transformation system of Agrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenic soil-dwelling bacteria that genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry the genes responsible for genetic transformation of the plant [see, for example, Kado: Crit. Rev. Plant Sci. 10 1 (1991)]. Descriptions of Agrobacterium vector systems and Agrobacterium-mediated gene transfer methods can be found in numerous publications. See, for example, Horsch et al.: Science 227 1229 (1985); Hoekema et al.: Nature 303 179 (1983); de Framond et al.: Bio/Technology 1 262 (1983); Jordan et al.: Plant Cell Reports 7 281 (1988); Leple et al.: Plant Cell Reports 11 137 (1992); Stomp et al.: Plant Physiol. 92 1226 (1990); Knauf et al.: Plasmid 8 45 (1982); Gruber et al.: supra; Miki et al.: supra; Moloney et al.: Plant Cell Reports 8 238 (1989); PCT Publication Nos. WO 84/02913, WO 84/02919 and WO84/02920; EP Patent No. 116,718; and U.S. Patent Nos. 4,940,838, 5,464,763 and 5,929,300.

A commonly used method for transforming plants is microprojectile-mediated transformation, where DNA is delivered on the surface of microprojectiles. The expression vector is delivered into plant tissues using a biolistic structure that accelerates the microprojectiles to the extent necessary to penetrate plant cell walls and membranes [Sanford et al.: Part. Sci. Technoi. 5 27 (1987), Sanford: Trends Biotech. 6 299 ·; :··. .· >·.

-16(1988); Sanford: Physiol. Plant 79 206 (1990); Klein et al.: Biotechnology 10 268 (1992); Klein et al.: Nature 327 70 (1987)].

Another method for physically introducing DNA into plants is sonication of target cells [Zhang et al.: Bio/Technology 9 996 (1991)]. Alternatively, liposome or spheroplast fusion has been used to introduce expression vectors into plants [Deshayes et al.: EMBO J. 4 2731 (1985); Christou et al.: Proc. Natl. Acad. Sci. USA 84 3962 (1987)]. Direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-omitine has also been reported [Hain et al.: Mol. Gen. Genet. 199 161 (1985) and Draper et al.: Plant Cell Physiol. 23 451 (1982)]. Electroporation of protoplasts or whole cells and tissues has also been reported [Donn et al.: in: Abstracts of Vllth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin et al.: Plant Cell 4 1495 (1992); Spencer et al.: Plant Mol. Biol. 24 51 (1994); and Fromm et al.: Proc. Natl. Acad. Sci. USA 82 5824 (1985)]. Other methods include microinjections [Crossway: Mol. Gen. Genetics, 202 179 (1985)], polyethylene glycol transformation [Krens et al.: Nature 296 72 (1982)], protoplast fusion with other entities, whether minicells, cells, lysosomes, or other fusible lipid-surfaced bodies [Fraley et al.: Proc. Natl. Acad. Sci. USA 79 1859 (1982); and the techniques disclosed in U.S. Patent No. 5,231,019].

After selection, the transformed plant cells are regenerated into transgenic plants. Plant regeneration methods are well known in the art and include those described in the following publications: Handbook of Plant Cell Culture, vols. 1-3, eds.: Evans et al., Macmillan Publishing Co., New York, N.Y. (1983, 1984, 1984, respectively); Predieri and Malavasi: Plant Cell, Tissue and Organ Culture 17 133 (1989); James et al.: J. Plant Physiol. 132 148 (1988); Fasolo et al.: Plant Cell, Tissue and Organ Culture 16 75 (1989); Valobra and James: Plant Cell, Tissue and Organ Culture 21 51 (1990); Srivastava et al.: Plant Science 42 209 (1985); Rowland and Ogden: Hort. Science 27 1127 (1992); Park and Son: Plant Cell, Tissue and Organ Culture 15 95 (1988); Noh and Minocha: Plant Cell Reports 5 464 (1986); Brand and Lineberger: Plant Science 57 173 (1988); Bozhkov et al.: Plant Cell Reports 11 386 (1992); Kvaalen and von Arnold: Plant Cell, Tissue and Organ Culture 27 49 (1991); Tremblay and Tremblay: Plant Cell, Tissue and Organ Culture 27 95 (1991); Gupta and Pullman: US Patent No. 5,036,007; Michler and Bauer: Plant Science 77 111 (1991); Wetzstein et al.: Plant Science 64 193 (1989); McGranahan et al.: Bio/Technology 6

- 17800 (1988); Gingas: Hort. Science 26 1217 (1991); Chalupa: Plant Cell Reports 9 398 (1990); Gingas and Lineberger: Plant Cell, Tissue and Organ Culture 17 191 (1989); Bureno et al.: Phys. Plant. 85 30 (1992); and Roberts et al.: Can. J. Bot. 68 1086 (1990).

Any type of transgenic plant can be produced according to the invention. Such plants include, for example, species from the following genera: Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Ceranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Permitetum, Ranunculus, Sencia, Salpiglossis, Cucumis, Browalia, Glycine, Lolium, Zea, Triticum, Sorghum, Malus, Apium, Datura and woody dicotyledonous forest-forming tree species. Specifically, broadleaf plants (dicotyledons, including beans, soybeans, cotton, peas, potatoes, sunflowers, tomatoes, tobacco, fruit trees, ornamentals, and trees) that are currently known to be damaged by auxin herbicides can be transformed to become resistant to these herbicides. Other plants (such as corn, sorghum, small grains, sugarcane, asparagus, and legumes) that are currently known to be resistant to auxin herbicides can also be transformed to become more resistant to these herbicides.

The invention also provides a method for controlling weeds in a field where transgenic plants are grown. The method comprises applying an effective amount of auxin herbicides to the field to control weeds. Methods for applying auxin herbicides and effective amounts for various weeds are well known. See, for example, Crop Protection Reference, 11th Edition, Chemical & Pharmaceutical Press, Inc., New York, NY, (1995). The invention provides, for the first time, transgenic plants that are tolerant to significantly higher levels of auxin herbicides than those commonly used in agriculture for weed control.

Example 1

Production of a synthetic, plant-optimized sequence encoding 2,4-D dioxygenase

The DNA sequence of a 2,4-D dioxygenase (often referred to as monooxygenase) gene isolated from Alcaligenes eutrophus was obtained from the GenBank sequence database (accession number: M16730). From this DNA sequence, the only open reading frame

J. <> s’

- The amino acid sequence of the protein encoded by 18(ORF) was determined (sequence number 1). A table reflecting the codon usage of dicot ORFs was obtained from a composite selection of randomly selected cDNA sequences from cotton, Arabidopsis and tobacco, collected from the GenBank database. Tables reflecting the codon usage of monocot ORFs were obtained from a random selection of maize cDNA sequences, also collected from the GenBank database. These are shown in Table 1 and Table 2 below. Using these plant-specific codon usage tables, the primary amino acid sequence of bacterial 2,4-D dioxygenase was converted into DNA coding sequences that reflected the codon preferences of dicots and monocots (sequence number 2 and 3, respectively).

Subsequently, both dicot and monocot synthetic plant-optimized 2,4-D dioxygenase ORFs (sequences identified as ID numbers 2 and 3) were used to construct 2,4-D dioxygenase genes that could be efficiently expressed in transgenic plants. These synthetic genes were designated SAD (synthetic gene adapted for dicots) and SAM (synthetic gene adapted for monocots), respectively, for the dicot and monocot variants. In order to produce a translatable transcript, after gene construction and integration into the plant genome, the alfalfa mosaic virus [AMV, Gallie et al.: Nucleic Acids Res. 15 8693 (1987)] A 5' untranslated leader sequence representing the 5' untranslated leader sequence from the 35S transcript was incorporated into the structure of the synthetic genes. In addition, a Cys Ala Gly coding marker sequence was added to the 3' end of the synthetic coding regions for each version of the synthetic gene. Finally, to simplify cloning, the designed sequences contained a HindIII specific overhang at the 5' end and a SalI specific overhang at the 3' end. The complete designed sequences of the synthetic parts of the SAD and SAM genes are given in SEQ ID NOS: 3 and 4.

For the construction of the designed synthetic portions of the SAD and SAM genes, each sequence was cut into overlapping oligonucleotides, twelve oligonucleotides for each strand, for a total of twenty-four oligonucleotides for each DNA sequence. A complete list of oligonucleotides used for the construction of the synthetic portions of the SAD and SAM genes is shown in Tables 3A, 3B, 4A, and 4B. Standard phosphoramidite chemical synthesis of the oligonucleotides was performed by Integrated DNA Technologies, Inc., (Coralville, Iowa). The assembly of the synthetic DNA molecules was performed using a procedure based on a protocol published by Sutton et al. in 1995 on the World Wide Web (www.epicentre.com) using AmpliligaseJ thermostable ligase (Epicentre Technologies Inc., Madison, WI). The oligonucleotides were ::· e <>

- 19were first phosphorylated using T4 polynucleotide kinase (Invitrogen Life Technologies, Carlsbad, CA) as a mixture of top and bottom strand oligonucleotides for each synthetic DNA construct. Each mixture contained the following: 10 pmol of each oligonucleotide, 70 mM Tris/HCl pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol (DTT), 0.1 mM ATP, and 10 units of T4 polynucleotide kinase, in a total volume of 25 μΐ. Phosphorylation was achieved by incubating the mixtures at 37 oC for 30 min followed by a 10 min denaturing incubation at 70 oC. To anneal and ligate the oligonucleotides, each reaction mixture was treated in a thermocycler at 70 oC for 10 min and then cooled to 65 oC for 10 min. To each mixture, 65 μΐ water, 10 μΐ 10x Ampliligase buffer (Epicentre Technologies), and 2 μΐ Ampliligase (5 units/μΐ) were added sequentially, and the temperature was reduced to 40 oC for three hours.

In this phase, to increase the cloning efficiency, the complete synthetic DNA sequences of SAD and SAM were recovered from the appropriate annealing/ligation reaction by polymerase chain reaction (PCR) in an MJ Research Inc. (Waltham, MA) Model PTC-100 Thermocycler using Amplitaq GoldTM DNA polymerase following the instructions provided by the manufacturer, Perkin Elmer Life Sciences (Boston, MA). The PCR primers used to recover each sequence were: AGA TCC TTT TTA TTT TTA ATT TTC TTT C (sequence ID 6), a 28-mer representing the 5' end of the AMV leader sequence, which was used in both the SAD and SAM recovery PCR reactions, CTC CAG CAC ACT AAA CAA CAG CGT C (sequence ID 7), a 25-mer specific for the 3' end of the SAD sequence, and CTC CAG CAG CAC ACT AC A CCA CC (sequence ID 8), a 20-mer specific for the 3' end of the SAM sequence. PCR fragments of approximately 918 bp in size were subjected to gel purification according to Ausubel et al. [Ausubel et al.: Current Protocols in Molecular Biology, Green/Wiley Interscience, New York, (1989)] and were cloned between two XcmI restriction sites in plasmid pUCR19, a modified pUC19 vector designed for rapid cloning of PCR fragments using T-overlaps generated by XcmI digestion [described in O’Mahony and Oliver: Plant Molecular Biology 39 809 (1999)], to generate plasmids pUCRsynSAD and pUCRsynSAM. Following cloning, the inserts were sequenced to verify the sequence integrity of the designed synthetic portions of the SAD and SAM genes. DNA sequencing was performed using the dRhodamine Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA) »·« * ·< * ·*

-20 was performed according to the manufacturer's instructions. Sequencing reactions were analyzed using a Perkin Elmer/ABI Prism 310 automated sequencer.

Table 1: Codon usage of dicots

amino acid codon usage % amino acid codon usage % alanine GCU 45.10 lysine AAG 55.14 GCA 25.41 AAA 44.86 GCC 17.62 methionine AUG 100.00 GCG 11.87 phenylalanine UUC 51.60 argin AGA 32.71 WOO 48.40 AGG 23.18 proline cc 37.73 CGU 18.53 CCA 34.72 CGA 10.55 CCG 14.94 CGG 7.70 CCC 12.61 CGC 7.33 according to street 27.64 asp aragin AAC 51.76 UCA 19.32 AAU 48.24 AGU 15.84 aspartic acid GAU 65.56 AGC 13.98 GAC 34.44 UCC 13.50 cysteine UGU 56.16 UCG 9.72 UGC 43.84 threonine ACU 36.71 glutamic acid GAG 51.01 ACA 28.49 GAA 48.99 ACC 21.89 glutamine CAA 54.26 ACG 12.91 CAG 45.74 tryptophan UGG 100.00 glycine GGA 36.34 tyrosine UAC 52.04 GGU 35.49 WOW 47.96 GGC 14.14 valine GUU 40.71 GGG 14.03 GUG 26.01 histidine CAU 57.39 GUC 20.56 CAC 42.61 GUA 12.71 isoleucine AUU 43.44 stop codon UAA 41.50

amino acid codon usage % amino acid codon usage % AUC 36.28 UGA 40.73 AUA 20.28 UAG 17.77 leucine UUG 24.73 CUC 19.86 CUU 19.86 UUA 12.99 CAG 11.69 CUA 10.87

Table 2: Codon usage of monocots

amino acid codon usage % amino acid codon usage % alanine GCC 36.21 lysine AAG 79.42 GCG 24.24 AAA 20.58 GCU 23.10 methionine AUG 100.00 GCA 16.45 arginine AGG 27.35 uuuu 29.32 CGC 27.09 proline CCA 27.77 CGG 15.18 CCG 27.06 AGA 12.94 CCC 23.97 CGU 11.41 cc 21.20 CGA 6.03 according to AGC 24.00 asparagine AAC 68.33 street 23.41 AAU 31.67 street 15.47 aspartic acid GAC 62.72 UCG 13.97 GAU 37.28 UCA 13.59 cysteine UGC 73.56 AGU 9.57 UGU 26.44 threonine ACC 40.33 glutamic acid GAG 73.29 ACU 21.27 GAA 26.71 ACG 20.39 glutamine CAG 58.31 ACA 18.00 CAA 41.69 trip to the forest UGG 100.00

amino acid codon usage % amino acid codon usage % glycine GGC 41.40 tyrosine UAC 72.14 GGG 20.28 WOW 27.86 GGU 20.25 valine GUG 37.35 GGA 18.07 GUC 32.71 histidine CAC 63.83 GUU 22.11 CAU 36.17 GUA 7.83 isoleucine AUC 57.82 stop codon UGA 44.57 AUU 28.52 UAG 28.24 AUA 136.5 UAA 27.18 leucine CUC 27.01 CUU 27.01 CUG 23.80 UUG 11.90 CUA 6.23 UUA 4.05

Table 3A: Dicotyledonous oligonucleotides, sense strand

AGCTAGATCCTTTTTATTTTTAATTTCTTTTCAAATACTTC CAG 13. sequence according to identification number ATCCATGTCTGTTGTTGCTAACCCTTTGCATCCTTTGTTCG CTGCTGGAGTTGAGGATATTGATCTCAGAGAAGCATTGG 14. sequence according to identification number GTTCTACTGAGGTGAGAGAAATTGAGAGACTCATGGACG AAAAGTCAGTTCTCGTTTTCAGAGGTCAACCACTCTCACA G 15. sequence according to identification number GATCAACAGATTGCTTTTGCTAGGAATTTTGGACCTTTGG AGGGTGGATTCATCAAAGTGAACCAGAGACCATCTAGGT T 16. sequence according to identification number CAAATATGCTGAACTCGCTGATATCTCTAATGTTTCATTG GATGGTAAGGTGGCACAAAGAGACGCTAGAGAAGTTGTG G 17. sequence according to identification number GAAATTTTGCAAATCAATTGTGGCATTCTGATTCTTCATTC 18. identification number

-23 I·*···· ·· ·· ·· · · · · ·

CAACAGCCAGCAGCTAGATATTCTATGTTGTCAGCTGTT sequence according to GTTGTGCCTCCTTCTGGAGGTGATACAGAATTTTGTGATA TGAGGGCAGCTTACGATGCTCTCCCAAGGGATTTGCAGTC 19. sequence according to identification number TGAACTCGAGGGATTGAGAGCTGAACATTACGCTTTGAA CTCAAGATTTCTCTTGGGAGATACTGATTACTCAGAGGCA C 20. sequence according to identification number AGAGAAACGCTATGCCTCCTGTTAACTGGCCTCTCGTTAG GACTCATGCTGGTTCTGGTAGAAAGTTCTTGTTTTATTGGA 21. sequence according to identification number GCACATGCTTCACATGTTGAGGGTCTCCCTGTTGCTGAGG GAAGAATGTTGCTCCGCTGAATTGCTCGAACATGCTACTCA 22. sequence according to identification number AAGAGAGTTTGTTTATAGACACAGATGGAATGTTGGTGA CTTGGTTATGTGGGATAATAGATGTGTGTTGCATAGAGGT THE 23. sequence according to identification number GGAGATATGATATTCTGCTAGAAGGGAACTCAGAAGGG CTACTACTTTGGATGACGCTGTTGTTTAGTGTGCTGGAG 24. sequence according to identification number

Table 3B: Dicotyledonous oligonucleotides, antisense strand

GAACAAAGGATGCAAAGGGTTAGCAACAACAGACATGGA TCTGGAAGTATTGAAAGAAAATTAAAAATAAAAAGGAT CT 25. sequence according to identification number TCGTCCATGAGTCTCTCAATTTCTCTCACCTCAGTAGAACC CAATGCTTCTCTGAGATCATATCCTCAACTCCAGCAGC 26. sequence according to identification number CCAAAGGTCCAAAATTCCTAGCAAAAGCAATCTGTTGATC CTGTGAGAGTGGTTGACCTCTGAAACGAGAACTGACTTT 27. sequence according to identification number CAATGAAACATTAGAGATATCAGCGAGTTCAGCATATTTG AACCTAGATGGTCTCTGGTTCACTTTGATGAATCCACCCT 28. sequence according to identification number AATGAAGAATCAGAATGCCACAATTGATTTGCAAAATTTC CCACAACTTCTCTAGCGTCTCTTGTGCCACCTTACCATC 29. sequence according to identification number TATCACAAAATTCTGTATCACCTCCAGAAGGAGGCACAAC AACAGCTGACAACATAGAATATCTAGCTGCTGGCTGTTGG 30. sequence according to identification number GTTCAAAGCGTAATGTTCAGCTTCCAATCCCTCGAGTTCA GACTGCAAATCCCTTGGGAGAGCATCGTAAGCTGCCCTCA 31. sequence according to identification number CTAACGAGAGGCCAGTTAACAGGAGGCATAGCGTTTCTCT 32. identification number

GTGCCTCTGAGTAATCAGTATCTCCCAAGAGAAATCTTGA sequence according to CCTCAGCAACAGGGAGACCCTCAACATGTGAAGCATGTG CTCCAATAAACAAGAACTTTCTACCAGAACCAGCATGAGT C 33. sequence according to identification number GTCACAACATTCCATCTGTGTCTATAAACAAACTCTCTTT GAGTAGCATGTTCGAGCAATTCAGCGAGCAACATTCTTC 34. sequence according to identification number GCCCTTCTGAGTTCCCTTCTAGCAGAAATATCATATCTCCT ACCTCTATGCAACACACATCTATTATCCCACATAACCAA 35. sequence according to identification number TCGACTCCAGCACACTAAACAACAGCGTCATCCAAAGTA GTA 36. sequence according to identification number

Table 4A: Monocot oligonucleotides, sense strand

AGCTAGATCCTTTTTTATTTTTAATTTTCTTTCAAATACTTCC BRANCH 37. sequence according to identification number ATCCATGTCCGTGGTGGCCAACCCACTCCACCCGCTCTTC GCGGCCGGCGTGGAGGATATCGACCTCAGGGAGGCGCTG G 38. sequence according to identification number GCAGCACCGAAGTGCGCGAAATCGAGAGGCTCATGGACG AGAAGAGCGTCCTCGTCTCCGCGGCCAACCACTCTCACA G 39. sequence according to identification number GATCAACAGATTGCTTTTGCTAGGAATTTTGGACCTTTGG AGGGTGGATTCATCAAGGTGAACCAGCCGCCCGTCCAGGTT 40. sequence according to identification number CAAGTACGCTGAACTGGCCGACATCAGCAACGTGTCCCTC GATGGGAAGGTGGCCCAGAGGGACGCTAGGGAAGTTGTG G 41. sequence according to identification number GCAACTTCGCCAACCAACTGTGGCACTCCGATAGCTCTTT CCAACAGCCAGCAGCCAGGTACTCCATGCTGAGCGCCGTC 42. sequence according to identification number GTCGTGCCACCATCCGGCGGTGACACCGAGTTCTGCGATA TGCGCGCCGCGTACGACGCCCTCCCGAGGGATCTGCAGA G 43. sequence according to identification number CGAGCTGGAGGGCCTCCGCGCGGAGCACTACGCCCTCAA CAGCAGGTTCCTCCTGGGGGACACTGACTACTCCGAGGCC C 44. sequence according to identification number

AGAGGAACGCGATGCCACCAGTGAACTGGCCCCTCGTCC GCACCCACGCTGGCAGCGGCCCGCAAGTTCCTGTTCATCGG G 45. sequence according to identification number GCCCATGCCTCCCATGTGGAGGGTCTCCCTGTCGCGGGAGG GCCGCATGCTCCTGGCCGAGCTCCTGGAGCACGCCACCCA 46. sequence according to identification number ACGCGAGTTCGTCTACCGCCACAGGTGGAATGTCGGCGAC CTCGTCATGTGGGATAACCGCTGCGTGCTGCACCGCGGCA 47. sequence according to identification number GGCGCTACGATATCAGCGCGCGCAGGGAACTCAGGCGCG CCACCACCCTCGACGACGCGGTGGTGTAGTGTGCTGGAG 48. sequence according to identification number

Table 4B: Monocot oligonucleotides, antisense strand

GAAGAGCGGGTGGAGTGGGTTGGCCACCACGGACATGGA TCTGGAAGTATTGAAAGAAAATTAAAAATAAAAAGGAT CT 49. sequence according to identification number TCGTCCATGAGCCTCTCGATTTCCGGCACTTCGGTGCTGCC CAGCGCCTCCCTGAGGTCGATATCCTCCACGCCGGCCGC 50. sequence according to identification number CCAAAGGTCCAAAATTCCTAGCAAAAGCAATCTGTTGATC CTGTGAGAGTGGTTGGCCGCGGGAAGACGAGGACGCTCTTTC 51. sequence according to identification number GAGGGACACGTTGCTGATGTCGGCCAGTTCAGCGTACTTG AACCTGGACGGGCGCTGGTTCACCTTGATGAATCCACCCT 52. sequence according to identification number AAAGAGCTATCGGAGTGCCACAGTTGGTTGGCGAAGTTGC CCACAACTTCCCTAGCGTCCCTCTGGGCCACCTTCCCCATC 53. sequence according to identification number TATCGCAGAACTCGGTGTCACCGCCGGATGGTGGCACGAC GACGGCGCTCAGCATGGAGTACCTGGCTGCTGGCTGTTGG 54. sequence according to identification number GTTGAGGGCGTAGTGCTCCGCGCGGAGGCCCTCCAGCTCG CTCTGCAGATCCCTCGGGAGGGCGTCGTACGCGGCGCGCA 55. sequence according to identification number CGGACGAGGGGCCAGTTCACTGGTGGCATCGCGTTCCTCT GGGCCTCGGAGTAGTCAGTGTCCCCCAGGAGGAACCTGCT 56. sequence according to identification number CCTCCGCGACAGGGAGACCCTCCACATGGGAGGCATGGG CCCCGATGACAGGAACTTTGCGGCCGCTGCCAGCGTGGG TG 57. sequence according to identification number GTCGCCGACATTCCACCTGTGGCGGTAGACGAACTGCGCGT TGGGTGGCGTGCTCCAGGAGCTCGGCCAGGAGCATGCGG 58. sequence according to identification number

-26J. ·“· ·· *·?

c GCGCGCCTGAGTTCCCTGCGCGCGCTGATATCGTAGCGCC TGCCGCGGTGCAGCACGCAGGCGGTTATCCCACATGACGA G 59. sequence according to identification number TCGACTCCAGCACACTACACCACCGCGTCGTCGAGGGTGG TG 60. sequence according to identification number

Example 2

Engineering a plant-expressible SAD gene

To generate a complete and plant competent SAD gene, the synthetic parts of the SAD gene carried in the pUCRsynSAD plasmid were removed by first releasing the 5' end of the synthetic sequence by XbaI digestion and removing the overhang using DNA polymerase I (Klenow large fragment) followed by KpnI digestion. This fragment was ligated into the pProPCISV plasmid, a pUC19 plasmid containing an amplified peanut chlorotic streak virus (PC1SV) promoter from plasmid pKLP36 [described by Maiti and Shepherd, Maiti and Shepherd: Biochem. Biophys. Res. Com. 244 440 (1998)], by first digesting with NcoI, treating with the addition of DNA polymerase I (Klenow large fragment) to fill in the created overhang and then digesting with KpnI. This created the plasmid pProPCISV-SAD, in which the synthetic part of the SAD gene, including the 5’ AMV leader and 3’ region encoding the Cys Ala Gly signature, was directly linked to the 3’ end of the PC1SV promoter (Figure 1). This plasmid served as the source for the PC1SV-SAD construct for insertion into binary vectors for the final gene construct before introduction into Agrobacterium for plant transformation.

Two binary vectors were selected for the final SAD gene construction, plasmids pPZP211PNPT-311g7 (Figure 2) and pPZP211-PNPT-512g7 (Figure 3). These two vectors are based on the pPZP family of vectors described by Hajdukiewicz et al. [Hajdukiewicz et al.: Plant Molec. Biol. 25 989 (1994)] and are pPZP211 derivatives in which the neomycin phosphotransferase II (NPTII) gene for kanamycin resistance is under the control of the PC1SV promoter and the g7 polyA termination sequence is located adjacent to a multicloning site (MCS, Figures 2 and 3). The only difference between these two vectors is the position of the MCS relative to the g7 polyA termination sequence. The g7 polyA termination sequence is the 3’ polyA termination signal from gene 7 of the octopine Agrobacterium tume,··. ......:

: .··. :··. ·' ·**· ·«.··« ·· · ·

-27 faciens TI plasmid within the T-bal region of octopin and as an EcoRI-Sal fragment isolated from plasmid pAP2034 [Velten and Schell: Nucleic Acids 13 6981 (1985)].

The complete SAD gene was constructed by removing the PC1SV-SAD sequence from the pProPCISV-SAD plasmid as a HindIII-SmaI fragment and inserting it into both pPZP21 l-PNPT-31 lg7 and pPZP21 l-PNPT-512g7 plasmids, which were first cleaved with BamHI, treated with the addition of DNA polymerase I (Klenow large fragment) to fill in the overhanging sequence, and then digested with HindIII. These reactions generated the following two vectors: pPZP21 l-PNPT-311-SAD (Figure 4) and pPZP211-PNPT-512-SAD (Figure 5), which contained the complete plant expressible SAD gene in one or two orientations relative to the PC1SV-NPTII-35SpoliA construct. This arrangement was used for the insertion of the SAD gene into plant genomes because the effect of the presence of two PC1SV promoter sequences within the same plasmid on both transformation and efficient transfer of the expressed trait was uncertain. By inserting the SAD gene into the vectors in such a way that the PC1SV promoters were inserted as both direct and inverted repeats, the possibility of a negative outcome was avoided.

After construction, the SAD genes in each vector were sequenced as described above to ensure sequence fidelity. This sequencing revealed that during the construction of pProPClSVSAD, an out-of-frame ATG codon was inserted into the 5’ untranslated leader sequence. The presence of this ATG codon could alter the translatability of the transcript that would be synthesized from the SAD gene and was therefore removed by PCR mutagenesis to restore the normal AMV leader sequence. Following repair, the sequence was re-checked for sequence fidelity. The original SAD gene, which contained the out-of-frame ATG codon, was designated SAD1 (as some transformation experiments using this construct have already begun). The improved SAD gene is referred to as the SAD2 gene and is the only version of the gene that was used to integrate the SAD construct into the cotton genome.

Example 3

Introducing SAD2 into cotton

The binary vectors containing the SAD2 gene, pPZP211-PNPT-311-SAD2 and pPZP211-PNPT-512-SAD2, were introduced individually by direct transformation as described by Walker-Peach and Velten [Walker-Peach and Velten: Plant Molecular Biology Manual, Part Bl:1-19, eds.: Gelvin, Shilperoort and Verma, Kluwer Academic Publishers, Dordrecht, The Netherlands, (1994)].: .··. ......í

I · ·· · · * _# * *

-28 into Agrobacterium tumefaciens strain EHA 105 [Hood et al.: Transgenic Research 2 208 (1993)]. The constructs were then introduced into cotyledon explants from cotton variety Coker 312 (Coker Seeds Inc.) by Agrobacterium transfection. This was done by isolating sterile cotyledon tissues (from seedlings grown from surface sterilized seeds [Trolinder and Gooden: Plant Cell Reports 6 231 (1987)]), creating explants (using a sterile punch), and submerging the explants in a 48-hour EHA 105 culture grown at 28 oC containing the appropriate construct. The explants were then transferred to 2MST medium (MS medium + 0.2 mg/l 2,4-D and 0.1 mg/l kinetin) after removal of excess EHA 105. The infected cotyledon tissues were incubated on 2MST medium for two days at 28 oC and then transferred to TI + KCL medium (MS medium + 0.1 mg/l 2,4-D and 0.1 mg/l kinetin + 50 mg/l kanamycin sulfate and 250 mg/l cefotaxime). Once healthy callus tissue had formed, they were transferred to fresh TI + KCL medium (with 0.05 mg/l 2,4-D) for a second round of selection. After six weeks, somatic embryos were formed on the surviving callus and mature transgenic cotton plants were produced as described in the publication of Trolinder and Goodin [Trolinder and Goodin: Plant Cell Reports 6 231 (1987)].

A total of 111 kanamycin-resistant cotton seedlings were produced (44 copies were produced in the pPZP211-PNPT-311-SAD2 transformations and 67 copies were produced in the pPZP211-PNPT-512SAD2 transformations). Each plant was tested for the presence of the SAD synthetic coding sequence and the NPTII coding sequence using PCR to ensure the integrity of the inserted DNA. PCR was performed as described above. The primers used in this study were: GGA GTT GAG GAT ATT GAT CTC AGA GAA GCA TTG (SEQ ID NO: 9) and GCG ATC TGC TGA TCC TGA CTC (SEQ ID NO: 10) for the SAD coding region and CGT CAA GAA GGC GAT AGA AGG (SEQ ID NO: 11) and GCT ATG ACT GGG CAC AAC AGA C (SEQ ID NO: 12) for the NPTII coding region. Of the 44 pPZP211-PNPT-311-SAD seedlings that survived the kanamycin treatment, 2 were found to be negative for PCR assays. Of the 67 pPZP21-PNPT-512-SAD seedlings that survived the kanamycin treatment, 14 were found to be negative for PCR assays.

The remaining 95 plants were grown in pots in the greenhouse until at least the transplant stage (approximately 45 centimeters in height) and then sprayed with 2,4-D-amine (United Agri Products, Greeley, CO) at a rate of approximately 1 kg/hectare of acid equivalent. This is twice the normal field rate for 2,4-D application. The 95 plants were: .··. ......í J. ·’> L?

-29zül 22 survived this treatment with little or no herbicidal damage observed. Eleven of these plants contained the pPZP211-pPZP211-PNPT-311-SAD2 construct and 11 contained the pPZP21 l-PNPT-512SAD2 construct. Each plant was grown to maturity in the greenhouse.

Only 7 of the 22 transgenic plants produced seeds. The remaining plants were presumably infertile. This infertileness was presumably an effect of the regeneration process, which is common in cotton. Of the seven fertilized plants, 3 contained the pPZP211-PNPT-311-SAD2 construct and 4 contained the pPZP211-PNPT-512-SAD2 construct.

To confirm that the inserted synthetic SAD genes were not heritable and to gain insight into the number of gene insertions, seeds from seven fertile SAD transgenic cotton plants were planted in hydroponic rockwool blocks (Hummert, St. Louis, MO) soaked in Peters Professional Water-Soluble Nutrient (5-11-26 HYDRO-SOL, supplemented with calcium nitrate and magnesium sulfate to provide complete nutrient supply, Hummert, St. Louis, MO). Hydroponic rockwool blocks were placed on tables in the greenhouse and nutrients were maintained at optimal levels using a non-recirculating hydroponic irrigation system. Plants were grown under greenhouse conditions (28 oC ± 5 oC air temperature) for 24 days. At this point, the plants were transferred to a spray booth, sprayed with 2,4-D amine at the normal field rate, i.e. 0.5 kg/hectare acid equivalent, kept in the spray booth for 24 hours to allow the 2,4-D to evaporate and then returned to the greenhouse. The effect of the treatment was visually assessed after 10-14 days and the results are presented in Table 5 below.

Table 5

transgenic line number of plants no damage minor damage serious damage ratio copper / nickel 311-22-1-4 43 16 16 11 3:1 311-22-1-5 47 6 18 23 1:1 311-22-1-8 56 14 28 14 3:1 512-21-1-5 56 8 34 14 3:1 512-21-4-3 30 7 20 3 9:1 512-21-4-5 41 5 24 12 2:1 512-24-4-4 63 7 21 35 1:1

-30• ** ·· ·· · ·

2,4-D copper control 55 7 33 15 3:1 2,4-D-free control 45 0 20 25 1:1

2,4-D copper control = transgenic 2,4-D resistant cotton containing the naturally occurring tfdA gene construct. [Bayley et al.: Theoretical Applied Genetics 83 645 (1992)]

2,4-D sense control = Coker 312 (non-transgenic) somatic embryos regenerated in the same manner as those containing SAD constructs _ slight damage = slight leaf wilting and minimal leaf desiccation severe damage = all leaves wilted and desiccation damage evident

The res/sens ratio was calculated by dividing the number of plants that showed some degree of resistance to 2,4-D treatment during the experiment by the total number of plants that showed severe damage or death. The negative control Cooker 312, regenerated from tissue culture, showed some degree of resistance, so these ratios cannot be considered a definitive measure of the Mendelian inheritance of the SAD gene. However, all negative control plants showed 2,4-D-induced damage, while all transgenic lines containing the SAD gene contained individuals that showed no damage at all.

From each of the 7 lines that showed no damage when treated with 2,4-D 24 days after germination, five plants were selected and individual samples of newly emerged leaves - one sample per plant - were taken for PCR testing, which was performed as described above. All plants were found positive for the SAD construct by PCR. These plants were grown for an additional 14 days and then re-sprayed with 2,4-D amine at the normal field rate, i.e. 0.5 kg/hectare acid equivalent. All 35 plants showed no damage after this treatment, while no negative control survived this spray. The 35 plants were grown to maturity and seeds were collected from them.

-31 .: . -3.....»-·'·

J. L n?

All references cited throughout this specification, including publications, patents, and published patent applications, are incorporated herein by reference.

Translation of free texts in the sequence list

Sequence 2: Dicotyledon open reading frame (ORF) for 2,4-D degradation

Sequence 3: Monocot open reading frame (ORF) for 2,4-D degradation

Sequence 4: Dicotyledonous gene for 2,4-D degradation

Sequence 5: Monocot gene for 2,4-D degradation

Sequences 6-12: primer

Sequences 13-60: gene fragment

Claims (50)

.: .··. '* ”·ι .”· -32PATENT CLAIMS 1. A DNA molecule comprising a synthetic DNA sequence, wherein the synthetic DNA sequence encodes an enzyme that degrades 2,4-dichlorophenoxyacetic acid to dichlorophenol, and the synthetic DNA sequence comprises a naturally occurring microbial sequence encoding the enzyme, wherein at least a plurality of codons of the naturally occurring microbial sequence are replaced by codons preferred by a plant. 2. The DNA molecule of claim 1, wherein all codons of the naturally occurring microbial sequence that are least preferred by the plant are replaced by codons preferred by the plant. 3. The DNA molecule of claim 1, wherein at least 50% of the codons of the naturally occurring microbial sequence are replaced with codons preferred by the plant. 4. The DNA molecule of claim 3, wherein at least 80% of the codons of the naturally occurring microbial sequence are replaced with codons preferred by the plant. 5. A DNA molecule according to any one of claims 1-4, which comprises a naturally occurring bacterial sequence as a naturally occurring microbial sequence. 6. The DNA molecule according to any one of claims 1-4, wherein the codons of the naturally occurring microbial sequence are replaced by codons preferred by a dicotyledonous plant. 7. The DNA molecule of claim 6, comprising the nucleotide sequence shown as SEQ ID NO:2. 8. The DNA molecule of any one of claims 1-4, wherein the codons of the naturally occurring microbial sequence are replaced by codons preferred by a monocot plant. 9. The DNA molecule of claim 8, comprising the nucleotide sequence shown as SEQ ID NO:3. 10. A DNA construct comprising a synthetic DNA sequence, wherein: the synthetic DNA sequence is operably linked to sequences that regulate plant gene expression; the synthetic DNA sequence encodes an enzyme that degrades 2,4-dichlorophenoxyacetic acid to dichlorophenol; and -33a synthetic DNA sequence comprises a naturally occurring microbial sequence encoding an enzyme in which at least a plurality of codons of the naturally occurring microbial sequence are replaced with codons preferred by a plant. 11. The DNA construct of claim 10, wherein all codons of the naturally occurring microbial sequence that are least preferred by the plant are replaced by codons preferred by the plant. 12. The DNA construct of claim 10, wherein at least 50% of the codons of the naturally occurring microbial sequence are replaced with codons preferred by the plant. 13. The DNA construct of claim 12, wherein at least 80% of the codons of the naturally occurring microbial sequence are replaced with codons preferred by the plant. 14. The DNA construct according to any one of claims 10-13, wherein the naturally occurring microbial sequence comprises a naturally occurring bacterial sequence. 15. The DNA construct of any one of claims 10-13, wherein the codons of the naturally occurring microbial sequence are replaced by codons preferred by a dicotyledonous plant. 16. The DNA construct of claim 15, comprising the nucleotide sequence shown as SEQ ID NO:2. 17. The DNA construct of claim 15, comprising the nucleotide sequence set forth in SEQ ID NO:4. 18. The DNA construct of any one of claims 10-13, wherein the codons of the naturally occurring microbial sequence are replaced by codons preferred by a monocot plant. 19. The DNA construct of claim 18, comprising the nucleotide sequence set forth in SEQ ID NO:3. 20. The DNA construct of claim 18, comprising the nucleotide sequence set forth in SEQ ID NO:5. 21. The DNA construct of claim 10, which is a vector. 22. The DNA construct of claim 10, which is a plasmid. 23. The DNA construct of claim 10, which is apProPCISV-SAD plasmid. 24. The DNA construct of claim 10, which is plasmid pPZP211-PNPT-311-SAD. 25. The DNA construct of claim 10, which is plasmid pPZP211-PNPT-512-SAD. .: .··. _: χ -,» <;· J. ·''· ·'? : 26. The DNA construct of claim 10, wherein the expression control sequences comprise a peanut chlorotic streak virus promoter. 27. A transgenic plant or plant part containing a synthetic DNA sequence, in which: the synthetic DNA sequence is operably linked to sequences that regulate plant gene expression; the synthetic DNA sequence encodes an enzyme that degrades 2,4-dichlorophenoxyacetic acid to dichlorophenol; and the synthetic DNA sequence comprises a naturally occurring microbial sequence encoding an enzyme in which at least a plurality of codons of the naturally occurring microbial sequence are replaced with codons preferred by the plant. 28. The plant or plant part of claim 27, wherein the expression control sequences comprise a peanut chlorotic streak virus promoter. 29. The plant or plant part of claim 27, wherein all codons of the naturally occurring microbial sequence that are least preferred by the plant are replaced by codons preferred by the plant. 30. The plant or plant part of claim 27, wherein at least 50% of the codons of the naturally occurring microbial sequence are replaced with codons preferred by the plant. 31. The plant or plant part of claim 30, wherein at least 80% of the codons of the naturally occurring microbial sequence are replaced with codons preferred by the plant. 32. A plant or plant part according to any one of claims 27-31, comprising a naturally occurring bacterial sequence as a naturally occurring microbial sequence. 33. The plant or plant part of claim 27, which is a dicotyledonous plant or plant part, and wherein the codons of the naturally occurring microbial sequence are replaced with codons preferred by a dicotyledonous plant. 34. The plant or plant part of claim 33, wherein one or more cells comprise the nucleotide sequence of SEQ ID NO:2. 35. The plant or plant part of claim 33, wherein one or more cells comprise the nucleotide sequence of SEQ ID NO:4. 36. The plant or plant part of claim 27, which is a monocot plant or plant part, and wherein the codons of the naturally occurring microbial sequence are replaced with codons preferred by a monocot plant. 37. The plant or plant part of claim 36, wherein one or more cells comprise the nucleotide sequence of SEQ ID NO:3. 38. The plant or plant part of claim 36, wherein one or more cells comprise the nucleotide sequence of SEQ ID NO:5. 39. The plant or plant part of claim 27, which is a cotton plant or part thereof. 40. The plant part of claim 27, which is a seed. 41. The plant part of claim 27, which is a fruit. 42. Method for controlling weeds in open ground containing a transgenic cultivated plant, characterized in that: an amount of auxin herbicide is applied to the field that is effective in controlling weeds in the field; the cultivated transgenic plant is resistant to the auxin herbicide as a result of containing a synthetic DNA sequence; the synthetic DNA sequence is operably linked to sequences that regulate plant gene expression; the synthetic DNA sequence encodes an enzyme that degrades 2,4-dichlorophenoxyacetic acid to dichlorophenol; and _: the synthetic DNA sequence comprises a naturally occurring microbial sequence encoding an enzyme in which at least a plurality of codons of the naturally occurring microbial sequence are replaced with codons preferred by the plant. 43. The method of claim 42, wherein the auxin herbicide is 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D-amine. 44. The method of claim 42, wherein a monocotyledonous plant is grown. 45. The method of claim 42, wherein a dicotyledonous plant is grown. 46. The method of claim 42, wherein a cotton plant is grown. 47. A method for selecting transformed plant cells, characterized in that: we provide a plant cell population; At least some of the plant cells in the population are transformed into a 10. with a DNA construct according to claim 1; and selecting the transformed plant cells by culturing the resulting population of plant cells in a culture medium containing an auxin herbicide, wherein the concentration of the herbicide is selected such that the transformed plant cells proliferate while the non-transformed plant cells do not proliferate. 5 48. The method of claim 47, wherein the auxin herbicide is 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D-amine. 49. A method for selecting transformed plants, characterized in that: providing a plant population that may include one or more plants comprising the DNA construct of claim 10; and) selecting transformed plants by applying a desired amount of an auxin herbicide to the plant population, wherein the amount of herbicide is selected such that the transformed plants grow while the growth of non-transformed plants is inhibited. 50. The method of claim 49, wherein the auxin herbicide is 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D-amine. The authorized representative: DANUBIA Patent and Trademark Office Ltd. patent attorney