EP1521833A2 - Gene synthetique de resistance a un herbicicde - Google Patents

Gene synthetique de resistance a un herbicicde

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Publication number
EP1521833A2
EP1521833A2 EP02793817A EP02793817A EP1521833A2 EP 1521833 A2 EP1521833 A2 EP 1521833A2 EP 02793817 A EP02793817 A EP 02793817A EP 02793817 A EP02793817 A EP 02793817A EP 1521833 A2 EP1521833 A2 EP 1521833A2
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EP
European Patent Office
Prior art keywords
plant
sequence
codons
dna
preferred
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP02793817A
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German (de)
English (en)
Inventor
Melvin J. Oliver
John J. Burke
Jeffrey P. Velten
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US Department of Agriculture USDA
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US Department of Agriculture USDA
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Application filed by US Department of Agriculture USDA filed Critical US Department of Agriculture USDA
Priority claimed from PCT/US2002/034084 external-priority patent/WO2003034813A2/fr
Publication of EP1521833A2 publication Critical patent/EP1521833A2/fr
Withdrawn legal-status Critical Current

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)

Definitions

  • the present invention relates to a synthetic herbicide-resistance gene, its use to prepare herbicide-resistant transgenic plants and its use as a selection marker.
  • 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 which encodes the first enzyme in the A . eutrophus 2,4-D degradation pathway is tfdA .
  • This gene encodes a dioxygenase which 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 plants, cotton plants, and hardwood trees containing the tfdA gene have been reported to have increased tolerance to 2,4-D. Streber et al .
  • the invention provides 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 natural microbial sequence that encodes the enzyme in which at least a plurality of the codons of the natural microbial sequence have been replaced by codons more preferred by a plant .
  • the invention also provides a DNA construct comprising the synthetic DNA sequence just described.
  • the synthetic DNA sequence is operatively linked to plant gene expression control sequences.
  • the invention further provides a transgenic plant or part of a plant.
  • the transgenic plant or plant part comprises the synthetic DNA sequence operatively linked to plant gene expression control sequences .
  • the invention also provides a method of controlling weeds in a field containing transgenic plants according to the invention.
  • the method comprises applying an amount of an auxin herbicide to the field effective to control the weeds in the field.
  • the transgenic plants are tolerant to the auxin herbicide as a result of comprising and expressing the synthetic DNA sequence. Indeed, for the first time, transgenic plants have been produced which are tolerant to levels of auxin herbicides substantially greater than those normally used in agriculture for controlling weeds.
  • the invention further provides methods of selecting transformed plants and plant cells.
  • the method of selecting transformed plant cells comprises providing a population of plant cells. At least some of the plant cells in the population are transformed with the DNA construct of the invention. Then, the resulting population of plant cells is grown in a culture medium containing an auxin herbicide at a concentration selected so that transformed plant cells proliferate and untransformed plant cells dol not proliferae .
  • the method of selecting transformed plants comprises providing a population of plants suspected of comprising a transgenic plant according to the invention. Then, an auxin herbicide is applied to the population of plants, the amount of herbicide being selected so that transformed plants will grow and growth of untransformed plants will be inhibited.
  • FIG. 1 Diagram of pProPClSV-SAD.
  • FIG. 1 Diagram of pPZP211-PNPT-311g7.
  • FIG. 1 Diagram of pPZP211-PNPT-512g7.
  • SAD 2 , 4-D-degrading synthetic gene adapted for dicots
  • CDS coding sequence
  • AMV-Leader 5' untranslated leader sequence from the 35S transcript of alfalfa mosaic virus
  • PC1SV- Promoter peanut chlorotic streak virus promoter
  • T-Left T-DNA left border from Agrobacterium tumefaciens nopaline Ti plasmid pTiT37
  • 35SPolyA 3' polyadenylation (polyA) termination signal sequence from the cauliflower mosaic virus (CaMV) 35S transcript
  • NPTII neomycin phosphotransferase II
  • g7PolyA 3 1 polyA termination signal from gene 7 within the T-Left border of an A. tumefaciens octopine plasmid
  • MCS multiple cloning site
  • T-Right T-DNA right border from A . tumefaciens Ti plasmid pTi
  • the invention provides a synthetic DNA sequence.
  • Synthetic is used herein to mean that the DNA sequence is not a naturally- occurring sequence .
  • 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 natural microbial sequence that encodes the enzyme, in which at least a plurality of the codons of the natural microbial sequence have been replaced by codons more preferred by a plant .
  • a “natural microbial sequence” is the coding sequence of a naturally-occurring microbial gene that encodes an enzyme that can degrade 2,4-D to DCP.
  • the "natural microbial sequence” may be the coding sequence of a cDNA or genomic clone isolated from a microorganism, may be a chemically-synthesized DNA molecule having the same coding sequence as that of such a clone, or may be a combination of such sequences.
  • Multi-enzyme pathways for 2,4-D degradation have been demonstrated in several genera of bacteria. See, e . g. , Lyon et al., Plant Molec . Biol . , 13, 533-540 (1989), and references cited 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, which is often referred to as a monooxygenase, but which is now known to be a dioxygenase (see Fukumori et al . , J " . Bacteriol .
  • the natural microbial sequence may be the coding sequence of a cDNA or genomic clone encoding a tfdA dioxygenase.
  • Such clones and their isolation are described in Bayley et al . , Theor. Appl . Genet . , 83, 645-649 (1992), Lyon et al . , Plant Molec . Biol . , 13, 533-540 (1989), Streber et al . , J. Bacteriology, 169, 2950-2955 (1987), Perkins and Lurquin, J.
  • bacteria are capable of degrading 2,4-D, including strains of Acinetobacter, Achromobacter , Alcaligenes, Arthrobacter, Corynebacterium, Flavobacterium, Pseudomona and strains of Actinomycetes ( e . g. , Nocardia spp . and Streptomyces viridochromogenes) (see, e . g. , Llewellyn and Last, in Herbicide- Resistant Crops, Chapter 10 (Stephen O. Duke ed., CRC Press Inc. (1996)), Bayley et al . , Theor. Appl . Genet .
  • the natural microbial sequence may be fully or partially chemically synthesized.
  • a cDNA or genomic clone obtained as described in the previous paragraphs, is sequenced by methods well known in the art. See, e . g. , Maniatis et al . , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor, NY (1982) , Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor, NY (1989) .
  • a synthetic DNA sequence comprising the coding sequence of the cDNA or genomic clone can be fully or partially chemically synthesized using methods well known in the art. See, e . g.
  • DNA sequences may be synthesized by phosphoamidite chemistry in an automated DNA synthesizer.
  • sequence of the tfdA gene from A . eutrophus JMP134 is publically available (see Streber et al . , J. Bacteriology, 169, 2950-2955 (1987), U.S. Patents Nos.
  • eutrophus tfdA gene can also be fully or partially chemically synthesized.
  • the preferred natural microbial sequence is a natural bacterial sequence.
  • a "natural bacterial sequence” is the coding sequence of a naturally-occurring bacterial gene that encodes an enzyme that can degrade 2,4-D to DCP.
  • the "natural bacterial sequence” may be the coding sequence of a cDNA or genomic clone isolated from a bacterium, may be a chemically-synthesized DNA molecule having the same coding sequence as that of such a clone, or may be a combination of such sequences.
  • Most preferably the natural bacterial sequence is the coding sequence of a cDNA or genomic clone isolated from a strain of A . eutrophus, a chemically-synthesized DNA molecule having the same coding sequence as that of such a clone, or a combination of such sequences .
  • codons more preferred by a plant also referred to herein as “plant-preferred codons”
  • a "codon more preferred by a plant”or a “plant-preferred codon” is a codon which is used more frequently by a plant to encode a particular amino acid than is the microbial codon encoding that amino acid.
  • the plant-preferred codon is the codon used most frequently by the plant to encode the amino acid.
  • the plant codon usage may be that of plants in general, a class of plants (e . g. , dicotyledonous plants), a specific type of plant ( e . g.
  • codon usage or preferences of a plant or plants can be deduced by methods known in the art. See, e . g. , Maximizing Gene Expression, pages 225-85 (Reznikoff & Gold, eds., 1986), Perlak et al., Proc . Natl . Acad. Sci . USA, 88, 3324-3328 (1991), PCT WO 97/31115 , PCT WO 97/11086, EP 646643, EP 553494, and U.S. Patents Nos. 5,689,052, 5,567,862, 5,567,600, 5,552,299 and 5,017,692.
  • the codons used by the plant or plants to encode all of the different amino acids in a selection of proteins expressed by the plant or plants, preferably those proteins which are highly expressed, are tabulated. This can be done manually or using software designed for this purpose (see PCT application WO 97/11086) .
  • codons more preferred by the plant in which the synthetic DNA sequence will be expressed will improve expression as compared to use of the natural microbial sequence.
  • the published reports indicate that codon usage affects gene expression in plants at the level of mRNA stability and translational efficiency. See, e . g. , Perlak et al . , Proc . Natl . Acad. Sci . USA, 88, 3324-3328 (1991); Adang et al . , Plant Molec . Biol . , 21:1131-1145 (1993); Sutton et al . , Transgenic Res . , 1:228- 236 (1992) .
  • codons of the natural microbial sequence need to be changed to plant-preferred codons in order to obtain improved expression.
  • at least the codons least preferred by the plant are changed to plant-preferred codons.
  • Codons least preferred by the plant are those codons in the natural microbial sequence that are used least by the plant or plants in question to encode a particular amino acid.
  • Plant-preferred codons can be introduced into the natural microbial sequence by methods well known in the art. For instance, site-directed mutagenesis can be used. See Perlak et al . , Proc . Natl . Acad . Sci . USA, 88, 3324-3328 (1991). See also Maniatis et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor, NY (1982), Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor, NY (1989) . However, the plant- preferred codons are preferably introduced into the natural microbial sequence by chemically synthesizing the entire DNA sequence encoding the 2,4-D degrading enzyme.
  • chemical synthesis is highly preferred when a large number of microbial codons are replaced by plant-preferred codons.
  • chemical synthesis has a number of advantages. For instance, using chemical synthesis allows other changes to the sequence of the DNA molecule or its encoded protein to be made to, e . g. , optimize expression ( e . g. , eliminate mRNA secondary structures that interfere with transcription or translation, eliminate undesired potential polyadenylation sequences, and alter the A+T and G+C content) , add unique restriction sites at convenient points, delete protease cleavage sites, etc.
  • the synthetic DNA sequence having plant-preferred codons substituted for at least a plurality of microbial codons will encode the same amino acid sequence as the natural microbial sequence if these substitutions are the only differences in the sequence of the synthetic DNA sequence as compared to the natural microbial sequence.
  • the synthetic DNA sequence may comprise additional changes as compared to the natural microbial sequence.
  • the synthetic DNA sequence may encode an enzyme which degrades 2,4-D to DCP, but which has an altered amino acid sequence as compared to the enzyme encoded by the (unmutated) natural microbial sequence as a result of one or more substitutions, additions or deletions in the natural microbial sequence. Methods of making such substitutions, additions and deletions are well known in the art and are described above.
  • DNA constructs comprising the synthetic DNA sequence operatively linked to plant gene expression control sequences.
  • DNA constructs are defined herein to be constructed (non-naturally occurring) DNA molecules useful for introducing DNA into host cells, and the term includes chimeric genes, expression cassettes, and vectors.
  • operatively linked refers to the linking of DNA sequences (including the order of the sequences, the orientation of the sequences, and the relative spacing of the various sequences) in such a manner that the encoded protein is expressed.
  • Methods of operatively linking expression control sequences to coding sequences are well known in the art. See, e . g. , Maniatis et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor, NY (1982), Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor, NY (1989) .
  • “Expression control sequences” are DNA sequences involved in any way in the control of transcription or translation. Suitable expression control sequences and methods of making and using them are well known in the art.
  • the expression control sequences must include a promoter.
  • the promoter may be any DNA sequence which shows transcriptional activity in the chosen plant cells, plant parts, or plants.
  • the promoter may be inducible or constitutive. It may be naturally- occurring, may be composed of portions of various naturally- occurring promoters, or may be partially or totally synthetic.
  • Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley and Reynolds, Nucleic Acids Res . , 15, 2343-61 (1987). Also, the location of the promoter relative to the transcription start may be optimized. See, e.g., Roberts, et al . , Proc . Natl Acad. Sci . USA, 76, 760-4 (1979) . Many suitable promoters for use in plants are well known in the art .
  • suitable constitutive promoters for use in plants include: the promoters from plant viruses, such as the peanut chlorotic streak caulimovirus (PC1SV) promoter (U.S. Patent No.
  • PC1SV peanut chlorotic streak caulimovirus
  • Suitable inducible promoters for use in plants include: the promoter from the ACE1 system which responds to copper (Mett et al. PNAS 90:4567-4571 (1993)); the promoter of the maize In2 gene which responds to benzenesulfonamide herbicide safeners (Hershey et al., Mol . Gen . Genetics 227:229-237 (1991) and Gatz et al . , Mol . Gen . Genetics 243:32-38 (1994)), and the promoter of the Tet repressor from TnlO (Gatz et al . , Mol . Gen . Genet . 227:229-237 (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.
  • An exemplary inducible promoter of this type is the inducible promoter 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 application of a chimeric transcription activator, XVE, for use in an estrogen receptor- based inducible plant expression system activated by estradiol (Zuo et al., The Plant Journal , 24:265-273 (2000)).
  • Other inducible promoters for use in plants are described in EP 332104, PCT WO 93/21334 and PCT WO 97/06269.
  • promoters composed of portions of other promoters and partially or totally synthetic promoters can be used. See, e.g., Ni et al., Plant J. , 7:661-676 (1995) and PCT WO 95/14098 describing such promoters for use in plants.
  • the promoter may include, or be modified to include, one or more enhancer elements.
  • the promoter will include a plurality of enhancer elements. Promoters containing enhancer elements provide for higher levels of transcription as compared to promoters that do not include 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, 143-156 (1997)). See also PCT WO 96/23898 and Enhancers And Eukaryotic Expression (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1983) .
  • the coding sequences are preferably also operatively linked to a 3 ' untranslated sequence.
  • the 3' untranslated sequence will include a transcription termination sequence and a polyadenylation sequence.
  • the 3' untranslated region can be obtained from the flanking regions of genes from Agrobacterium, plant viruses, plants or other eukaryotes.
  • Suitable 3 ' untranslated sequences for use in plants include those of the 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 employed.
  • the 5' untranslated sequence is the portion of an mRNA which extends from the 5' CAP site to the translation initiation codon. This region of the mRNA is necessary for translation initiation in plants and plays a role in the regulation of gene expression. Suitable 5' untranslated regions for use in plants include those of alfalfa mosaic virus, cucumber mosaic virus coat protein gene, and tobacco mosaic virus.
  • the DNA construct may be a vector.
  • the vector may contain one or more replication systems which allow it to replicate in host cells. Self-replicating vectors include plasmids, cosmids and viral vectors.
  • the vector may be an integrating vector which allows the integration into the host cell's chromosome of the synthetic DNA sequence encoding the 2 , 4-D-degrading enzyme.
  • the vector desirably also has unique restriction sites for the insertion of DNA sequences. If a vector does not have unique restriction sites, it may be modified to introduce or eliminate restriction sites to make it more suitable for further manipulations.
  • the DNA constructs of the invention can be used to transform any type of plant cells (see below) .
  • a genetic marker must be used for selecting transformed plant cells ("a selection marker"). Selection markers typically allow transformed cells to be recovered by negative selection (i . e . , inhibiting growth of cells that do not contain the selection marker) or by screening for a product encoded by the selection marker.
  • the most commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (np Jj) gene, isolated from Tn5, 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) .
  • Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3 ' -adenyl transferase, and the bleomycin resistance determinant.
  • selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or bromoxynil.
  • herbicides such as glyphosate, glufosinate or bromoxynil.
  • Comai et al . Nature 317:741-744 (1985), Stalker et al . , Science 242:419-423 (1988), Hinchee et al . , Bio/Technology 6:915-922 (1988), Stalker et al . , J “ . Biol . Chem . 263:6310-6314 (1988), and Gordon-Kamm et al . , Plant Cell 2:603-618 (1990) .
  • selectable marker genes for plant transformation are not of bacterial origin. These genes include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3 -phosphate synthase, and plant acetolactate synthase. 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 154,204.
  • GUS ⁇ -glucuronidase
  • ⁇ -galactosidase ⁇ -galactosidase
  • luciferase luciferase
  • chloramphenicol acetyltransferase ⁇ -glucuronidase (GUS)
  • GUS ⁇ -galactosidase
  • luciferase luciferase
  • chloramphenicol acetyltransferase Jefferson, R.A., 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 .
  • GFP green fluorescent protein
  • auxin herbicide is used herein to refer 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) butryic acid, 2- (4- chlorophenoxy)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 available commercially. See Crop Protection Reference (Chemical & Pharmaceutical Press, Inc., New York, NY, 11th ed., 1995) .
  • the preferred auxin herbicides are 2,4-D and its salts (including amine salts) and esters.
  • “Tolerance” means that transformed plant cells are able to grow (survive, proliferate and regenerate into plants) when placed in culture medium containing a level of an auxin herbicide that prevents untransformed cells from doing so. "Tolerance” also means that transformed plants are able to grow after application of an amount of an auxin herbicide that inhibits the growth of untransformed plants.
  • Methods of selecting transformed plant cells are well known in the art. Briefly, at least some of the plant cells in a population of plant cells (e . g. , an explant or an embryonic suspension culture) are transformed with a DNA construct comprising the synthetic DNA sequence of the invention. The resulting population of plant cells is placed in culture medium containing an auxin herbicide at a concentration selected so that transformed plant cells will grow, whereas untransformed plant cells will not. Suitable concentrations of an auxin herbicide can be determined empirically as is known in the art.
  • this amount may further need to be an amount which inhibits adventitious shoot formation from untransformed plant cells and allows adventitious shoot formation from transformed plant cells, since this is apparently the case with the natural-occurring bacterial tfdA gene. See U.S. Patent No. 5,608,147 and PCT application WO 95/18862.
  • 2,4-D should be present in an amount ranging from about 0.001 mg/l to about 5 mg/l culture medium, preferably from about 0.01 mg/l to 0.2 mg/l culture medium.
  • an auxin herbicide is applied to a population of plants which may comprise one or more transgenic plants comprising a DNA construct of the invention providing for 2,4-D degradation.
  • the amount of the auxin herbicide is selected so that transformed plants will grow, and the growth of untransformed plants will be inhibited.
  • the level of inhibition must be sufficient so that transformed and untransformed plants can be readily distinguished (i.e., inhibition must be statistically significant).
  • Such amounts can be determined empirically as is known in the art. See also Crop Protection Reference (Chemical & Pharmaceutical Press, Inc., New York, NY, 11 th ed. , 1995).
  • Selection based on tolerance to an auxin herbicide can be used in the production of plants tolerant to 2,4-D and other auxin herbicides, in which case the use of another selection marker may not be necessary. Absence of a separate selection marker is advantageous since it minimizes the number of foreign genes expressed. Selection based on tolerance to an auxin herbicide can also be used in the production of transgenic plants that express other genes of interest. Many such genes are known and include genes coding for proteins of commercial value and genes that confer improved agronomic traits on plants (see, e.g., PCT WO 97/41228, the complete disclosure of which is incorporated herein by reference) .
  • the DNA constructs of the invention can be used to transform a variety of plant cells.
  • the synthetic DNA sequence coding for the 2 , 4-D-degrading enzyme and the selection marker may be on the same or different DNA constructs. Preferably, they are arranged on a single DNA construct as a transcription unit so that all of the coding sequences are expressed together.
  • the gene(s) of interest and the synthetic DNA sequence coding for the 2 , 4-D-degrading enzyme when tolerance to an auxin herbicide is being used as a selection marker, may be on the same or different DNA constructs. Such constructs are prepared in the same manner as described above .
  • Suitable host cells include plant cells of any kind (see below) .
  • the plant cell is one that does not normally degrade auxin herbicides.
  • the present invention can also be used to increase the level of degradation of auxin herbicides in plants that normally degrade such herbicides.
  • 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 so that they are able to degrade these herbicides, and progeny of such transformed plants, plant parts and plant cells.
  • 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 so that they are able to degrade more of these herbicides or to degrade them more efficiently, and progeny of such transformed plants, plant parts and plant cells.
  • Plant should be understood as referring to a unicellular organism or a multicellular differentiated organism capable of photosynthesis, including algae, angiosperms (monocots and dicots) , gymnosperms, bryophytes, ferns and fern allies.
  • Plant parts are parts of multicellular differentiated plants and include seeds, pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, explants, etc.
  • Plant cell should be understood as referring to the structural and physiological unit of multicellular plants.
  • the term “plant cell” refers to any cell that is a plant or is part of, or derived from, a plant.
  • Some examples of cells encompassed by the present invention include differentiated cells that are part of a living plant, differentiated cells in culture, undifferentiated cells in culture, and the cells of undifferentiated tissue such as callus or tumors .
  • Methods of transforming plant cells are well known in the art. For instance, numerous methods for plant transformation have been developed, 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, Glick, B.R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 67-88. In addition, vectors and in vi tro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al . , "Vectors for Plant Transformation” in Methods in Plant Molecular Biology and Biotechnology, Glick, B.R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 89-119.
  • a . tumefaciens and A . rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells.
  • the Ti and Ri plasmids of A . tumefaciens and A . rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C.I., Crit. J?ev. Plant . Sci . 10:1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by numerous references. See, for example, Horsch et al .
  • a generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles.
  • the expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds sufficient to penetrate plant cell walls and membranes.
  • Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al .
  • liposome or spheroplast fusion have been used to introduce expression vectors into plants.
  • Direct uptake of DNA into protoplasts using CaCl 2 precipitation, polyvinyl alcohol or poly-L-ornithine have also been reported. Hain et al., Mol . Gen . Genet . 199:161 (1985) and Draper et al . , Plant Cell Physiol . 23:451 (1982).
  • transformed plant cells are regenerated into transgenic plants.
  • Plant regeneration techniques are well known in the art and include those set forth in the Handbook of Plant Cell Cul ture, Volumes 1-3, Evans et al . , eds. Macmillan Publishing Co., New York, N.Y. (1983, 1984, 1984, respectively); Predieri and Malavasi, Plant Cell , Tissue, and Organ Cul ture 17:133-142 (1989) ; James, D. J., et al., J " . Plant Physiol . 132:148-154 (1988); Fasolo, F., et al .
  • Transgenic plants of any type may be produced according to the invention.
  • Such plants include, for example, species from the 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, Pennisetum, Ranunculus, Sencia, Salpiglossis, Cucumis, Browalia, Glycine, Lolium, Zea, Triticum, Sorghum, Malus , Apium, Datura and woody dicotyledonous forest tree species.
  • broadleaf plants including beans, soybeans, cotton, peas, potatoes, sunflowers, tomatoes, tobacco, fruit trees, ornamental plants and trees
  • auxin herbicides can be transformed so that they become tolerant to these herbicides.
  • Other plants such as corn, sorghum, small grains, sugarcane, asparagus, and grass
  • auxin herbicides can be transformed to increase their tolerance to these herbicides.
  • the invention provides a method of controlling weeds in a field where transgenic plants are growing.
  • the method comprises applying an effective amount of an auxin herbicide to the field to control the weeds.
  • auxin herbicides Methods of applying auxin herbicides and the amounts of them effective to control various types of weeds are known. See Crop Protection Reference (Chemical & Pharmaceutical Press, Inc., New York, NY, 11th ed., 1995) .
  • transgenic plants have been produced which are tolerant to levels of auxin herbicides substantially greater than those normally used in agriculture for controlling weeds.
  • the DNA sequence of a 2,4-D dioxygenase (also often referred to as a monooxygenase,- see above) gene isolated from Alcaligenes eutrophus was obtained from the sequence database GenBank (accession number M16730) . From this DNA sequence, the amino acid sequence of the protein coded for by the single open-reading frame (ORF) was determined [SEQ ID N0:1].
  • ORF single open-reading frame
  • a codon usage table reflecting monocotyledonous ORFs was derived from a random selection of cDNA sequences from maize, also extracted from the GenBank database. These are Tables 1 and 2 below. Using these plant-specific codon usage tables, the derived primary amino acid sequence of the bacterial 2,4-D dioxygenase was converted into DNA coding sequences that reflected the codon preferences of dicotyledonous and monocotyledonous plants [SEQ ID NOS: 2 and 3, respectively] . The synthetic plant-optimized 2,4-D dioxygenase ORFs [SEQ ID NOS : 2 and 3], both dicot and monocot, were then used to design 2,4-D dioxygenase genes capable of efficient expression in transgenic plants.
  • SAD Synthetic gene Adapted for Dicots
  • SAM Synthetic gene Adapted for Monocots
  • a 5' untranslated leader sequence representing the 5' untranslated leader sequence from the 35S transcript of alfalfa mosaic virus (AMV; Gallie et al., Nucleic Acids Res., 15:8693-8711 (1987)
  • AMV alfalfa mosaic virus
  • a signature sequence encoding Cys Ala Gly, was added to the 3' end of the synthetic coding regions for each version of the synthetic gene.
  • the designed sequences included a Hindlll-specific overhang at the 5' end and a Sail-specific overhang at the 3' end.
  • the complete designed sequences for the synthetic portions of the SAD and SAM genes are SEQ ID NOS : 4 and 5.
  • each sequence was dissected into overlapping oligonucleotides, twelve oligonucleotides for each of the two strands resulting in a total of twenty-four oligonucleotides for each DNA sequence.
  • a complete list of the oligonucleotides used to construct the synthetic portions of the SAD and SAM genes is given in Tables 3A, 3B, 4A, and 4B below.
  • the oligonucleotides were synthesized using standard phosphoramidite chemistry by Integrated DNA Technologies, Inc., Coralville, Iowa.
  • the synthetic DNA molecules were assembled using a procedure based upon the protocol described by Sutton et al .
  • Oligonucleotides were first phosphorylated using T4 polynucleotide kinase (Invitrogen Life Technologies, Carlsbad, CA) as mixtures of upper and lower strand oligonucleotides for each synthetic DNA construct.
  • Each mixture contained 10 pmoles of each oligonucleotide, 70 mM Tris/HCl pH 7.6, 10 mM MgCl 2 , 5 mM dithiothreitol (DTT) , 0.1 mM ATP, and 10 units of T4 polynucleotide kinase, for a total volume of 25 ⁇ l. Phosphorylation was achieved by incubation of the mixtures at 37°C for 30 minutes, followed by a denaturing incubation at 70°C for 10 minutes. To anneal and ligate the oligonucleotides, each reaction mixture was retreated at 70°C for 10 minutes in a thermocycler and subsequently cooled to 65°C over a 10-minute period.
  • PCR primers used for the recovery of each sequence were AGATCCTTTTTATTTTTAATTTTCTTTC [SEQ ID NO: 6], a 28mer representing the 5' end of the AMV leader sequence and used for both the SAD and SAM recovery PCR reactions, and CTCCAGCACACTAAACAACAGCGTC [SEQ ID NO: 7] , a 25mer specific for the 3' end of the SAD sequence, and CTCCAGCACACTACACCACC [SEQ ID NO:8], a 20mer specific for the 3' end of the SAM sequence.
  • PCR fragments corresponding to the appropriate size of 918 bp were gel purified as described in Ausubel et al .
  • DNA sequencing was performed by use of a dRhodamine Terminator Cycle Sequencing kit (PE Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Sequence reactions were analyzed using a Perkin Elmer/ABI Prism 310 automated sequencer.
  • EXAMPLE 2 Construction Of A Plant-Expressible SAD Gene.
  • the synthetic portions of the SAD gene contained in pUCRsynSAD were removed by first releasing the 5' end of the synthetic sequence by digestion with Xbal and filling in the overhang with DNA polymerase I (Klenow large fragment) followed by digestion with Kpnl .
  • This fragment was ligated into the plasmid pProPClSV, a pUC19 plasmid containing an enhanced Peanut Chlorotic Streak Virus (PC1SV) promoter derived from pKLP36 (described by Maiti and Shepherd, Biochem . Biophys . Res . Com.
  • PC1SV enhanced Peanut Chlorotic Streak Virus
  • pPZP211-PNPT-311g7 Figure 2
  • pPZP211-PNPT-512g7 Figure 3
  • NPTII neomycin phosphotransferase II
  • the g7 polyA termination sequence is the 3 ' polyA termination signal from gene 7 within the octopine T-Left region of an octopine Agrobacterium tumefaciens Ti plasmid and was isolated as an EcoRI- Sall fragment from pAP2034 (Velten and Schell, Nucleic Acids, 13:6981-6998 (1985) ) .
  • the complete SAD gene was constructed by removal of the PCISV-SAD sequence from pProPClSV-SAD as a Hindlll-Smal fragment and insertion into both pPZP211-PNPT-311g7 and pPZP211-PNPT-512g7 that were first cut with BamHI, treated with DNA polymerase I (Klenow large fragment) to fill in the overhanging sequence, and subsequently digested with Hindlll.
  • SAD genes in each vector were sequenced as described above to ensure fidelity.
  • This sequencing revealed that, in the construction of pProPClSV-SAD, an out-of-frame ATG codon was introduced 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 so was deleted by PCR mutagenesis to restore the normal AMV leader sequence.
  • SAD1 since some transformation experiments had begun using this construct
  • the repaired SAD gene is referred to as SAD2 and is the only version of the gene used for integration of the SAD construct into the cotton genome .
  • the two binary vectors containing the SAD2 gene, pPZP211-PNPT-311- SAD2 and pPZP211-PNPT-512-SAD2 were individually introduced into the EHA 105 strain of Agrobacterium tumefaciens (Hood et al . , Transgenic Research, 2:208-218 (1993)) by direct transformation as described by Walker-Peach and Velten, in Plant Molecular Biology Manual , section Bl:l-19 (Gelvin, Shilperoort and Verma, eds., Kluwer Academic Publishers, Dordrecht, The Netherlands, 1994)).
  • the constructs were subsequently introduced by Agrobacterium transfection into cotyledon explants from the cotton variety Coker 312 (Coker Seed Inc.). This was achieved by isolating sterile cotyledon tissue (derived from seedlings grown in culture from surface-sterilized seed as described by Trolinder and Gooden, Plant Cell Reports, 6:231-234 (1987)), generating explants (by use of a sterile hole punch) , and submerging the explants in a 48-hour-old culture of EHA 105, containing the appropriate construct, grown at 28°C The explants were then transferred onto 2MST medium (MS medium + 0.2 mg/L 2,4-D and 0.1 mg/L kinetin) subsequent to removal of excess EHA 105.
  • 2MST medium MS medium + 0.2 mg/L 2,4-D and 0.1 mg/L kinetin
  • the infected cotyledon tissues were incubated on the 2MST medium for 2 days at 28°C prior to transfer to Tl+KCL medium (MS medium + 0.1 mg/L 2,4-D and 0.1 mg/L kinetin + 50 mg/L kanamycin sulphate and 250 mg/L Cefotaxime) .
  • Tl+KCL medium MS medium + 0.1 mg/L 2,4-D and 0.1 mg/L kinetin + 50 mg/L kanamycin sulphate and 250 mg/L Cefotaxime
  • a total of 111 kanamycin-resistant cotton seedlings were generated (44 were generated in the pPZP211-PNPT-311-SAD2 transformations, and 67 in the pPZP211-PNPT-512-SAD2 transformations) .
  • Each plant was analyzed for the presence of the SAD synthetic coding sequence and the NPTII coding sequence by PCR to ensure the integrity of the inserted DNA. The PCR was performed as described above.
  • the primers used for this analysis were GGAGTTGAGGATATTGATCTCAGAGAAGCATTG [SEQ ID NO: 9] and GCGATCTGCTGATCCTGACTC [SEQ ID NO: 10] for the SAD coding region and CGTCAAGAAGGCGATAGAAGG [SEQ ID NO: 11] and GCTATGACTGGGCACAACAGAC [SEQ ID NO: 12] for the NPTII coding region.
  • SEQ ID NO: 9 GCGATCTGCTGATCCTGACTC
  • 2 were shown to be negative by the PCR testing.
  • the 67 pPZP211-PNPT- 512-SAD seedlings that survived kanamycin treatment 14 were negative in the PCR tests.
  • 2,4-D res control transgenic 2,4-D resistant cotton containing the naturally-occurring tfdA gene construct.
  • 2,4-D sens control Coker 312 (not transgenic) regenerated from somatic embryos in the same manner as those containing the SAD constructs
  • the ratio "Res"/Sens was calculated as the number of plants that showed some resistance to 2,4-D treatment during the experiment divided by the combined number of plants that showed severe damage or death.
  • the negative control of Coker 312 that had been regenerated from tissue culture did show some signs of resistance, so these ratios are not to be considered as definitive measures of Mendelian inheritance of the SAD gene. Nevertheless, all of the negative control plants did show 2, 4-D-induced damage, whereas all of the transgenic lines that contain the SAD gene had individuals that exhibited no damage at all.
  • gca caa aga gac get aga gaa gtt gtg gga aat ttt gca aat caa ttg 336
  • gag gat ate gac etc agg gag gcg ctg ggc age ace gaa gtg cgc gaa 96 Glu Asp He Asp Leu Arg Glu Ala Leu Gly Ser Thr Glu Val Arg Glu 20 25 30
  • gag agg etc atg gac gag aag age gtc etc gtc ttc cgc ggc caa 144 He Glu Arg Leu Met Asp Glu Lys Ser Val Leu Val Phe Arg Gly Gin 35 40 45
  • gcc cag agg gac get agg gaa gtt gtg ggc aac ttc gcc aac caa ctg 336 Ala Gin Arg Asp Ala Arg Glu Val Val Gly Asn Phe Ala Asn Gin Leu 100 105 110
  • primer ⁇ 400 12 gctatgactg ggcacaacag ac 22

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Abstract

L'invention concerne une molécule d'ADN, une construction d'ADN, une plante transgénique et une partie de plante transgénique comprenant une séquence d'ADN synthétique. La séquence d'ADN synthétique code pour une enzyme qui dégrade l'acide 2,4-dichlorophénoxyacétique (2,4-D) en dichlorophénol. La séquence d'ADN synthétique comprend une séquence microbienne naturelle, codant pour l'enzyme, dans laquelle au moins plusieurs codons de la séquence microbienne naturelle ont été remplacés par des codons qui sont plus facilement tolérés par une plante. L'invention concerne encore un procédé de lutte contre les mauvaises herbes dans un champ contenant des plantes transgéniques de l'invention par application au champ d'un herbicide à base d'auxine, tel que le 2,4-D. L'invention concerne aussi des procédés de sélection de plantes et de cellules de plantes, transformées par une construction d'ADN de l'invention, au moyen d'un herbicide à base d'auxine.
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