Herbicide resistance gene, compositions and methods
View Patent ↗The present disclosure provides methods, recombinant DNA molecules, recombinant host cells containing the DNA molecules, and transgenic and genetically engineered plant cells, plant tissue, seeds and plants which contain and express an herbicide resistant protoporphyrinogen oxidase such that they germinate from seed and grow in the presence of an amount of herbicide where the parent plant does not. Such plants are especially appropriate for use in agriculture or horticulture where herbicides are used to kill undesirable plants which might contaminate or compete with the transgenic plant of interest.
1. A method for producing a genetically engineered, non-transgenic, herbicide resistant or tolerant plant cell, said method comprising the steps of:
(a) introducing into a plant cell a recombinagenic oligonucleotide with a targeted mutation in the protoporphyrin oxidase (PPO) gene to produce plant cells with a mutant PPO gene that expresses a mutant PPO protein that is deleted at an amino acid position corresponding to Gly210 or Gly 211 in the Amaranthus tuberculatus PPO protein or at an analogous amino acid residue in a PPO homolog; and
(b) identifying a plant cell having a mutant PPO protein that has substantially the same catalytic activity as compared to a corresponding wild type PPO protein in the presence of herbicide except that said mutant PPO protein is not inhibited by the herbicide; and
whereby a non-transgenic herbicide resistant plant cell is produced.
2. The method of claim 1 , wherein said herbicide resistant PPO is resistant to inhibition by lactofen, (±)-2-ethoxy-1-methyl-2-oxoethyl 5-{2-chloro-4-(trifluoromethyl)phenoxy}-2-nitrobenzoate; acifluorfen, 5-{2-chloro-4-(trifluoromethyl)phenoxy}-2-nitrobezoic acid; its methyl ester; or oxyfluorfen, 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluorobenzene)}, oxidiazoles, (e.g., oxidiazon, 3-{2,4-dichloro-5-(1-methylethoxy)phenyl}-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2-(3H)-one), cyclic imides (e.g., S-23142, N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide; chlorophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide), phenyl pyrazoles (e.g. TNPP-ethyl, ethyl 2-{1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy}propionate; M&B 39279), pyridine derivatives (e.g. LS 82-556), phenopylate and O-phenylpyrrolidino- and piperidinocarbamate analogs thereof, and 3-phenyluracils of formula I
wherein R 1 is methyl or NH 2 ; R 2 is C 1 -C 2 -haloalkyl; R 3 is hydrogen or halogen; R 4 is halogen or cyano; R 5 is hydrogen, cyano, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy, C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl, C 3 -C 7 -cycloalkyl, C 3 -C 6 -alkenyl, C 3 -C 6 -alkynyl or benzyl which is unsubstituted or substituted by halogen or alkyl; and R 6 , R 7 independently of one another are hydrogen, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy, C 3 -C 6 -alkenyl, C 3 -C 6 -alkynyl, C 3 -C 7 -cycloalkyl, C 3 -C 7 -cycloalkenyl, phenyl or benzyl, where each of the 8 abovementioned substituents is unsubstituted or may be substituted by 1 to 6 halogen atoms and/or by one, two or three groups selected from: OH, NH 2 , CN, CONH 2 , C 1 -C 4 -alkoxy, C 1 -C 4 -haloalkoxy, C 1 -C 4 -alkylthio, C 1 -C 4 -haloalkylthio, C 1 -C 4 -alkylsulfonyl, C 1 -C 4 -haloalkylsulfonyl, C 1 -C 4 -alkylamino, di(C 1 -C 4 -alkyl)amino, formyl, C 1 -C 4 -alkylcarbonyl, C 1 -C 4 -alkoxycarbonyl, C 1 -C 4 -alkylaminocarbonyl, di(C 1 -C 4 -alkyl)aminocarbonyl, C 3 -C 7 -cycloalkyl, phenyl and benzyl; or R 6 , R 7 together with the nitrogen atom form a 3-, 4-, 5-, 6- or 7-membered saturated or unsaturated nitrogen heterocycle which may be substituted by 1 to 6 methyl groups and which may contain 1 or 2 further heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur as ring members.
3. The method according to claim 1 wherein the recombinagenic oligonucleotide is a mixed duplex nucleotide which contains a first homologous region which has a sequence identical to the sequence of at least 6 base pairs of the first fragment of the target PPO gene and a second homologous region which has a sequence identical to the sequence of at least 6 base pairs of a second fragment of the target PPO gene, and an intervening region which contains at least one nucleobase heterologous to the target PPO gene, which intervening region connects the first and second homologous region.
4. The method according to claim 1 in which the recombinagenic oligonucleotide is introduced by electroporation, biolistic transformation or polyethylene glycol precipitation.
5. The method of claim 1 , further comprising after step (b), (c) regenerating a plant having a mutated PPO gene from said plant cell, whereby a non-transgenic herbicide resistant plant is produced.
6. The method according to claim 1 in which the amino acid deletion is in a corn, wheat, rice, barley, soybean, cotton, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, pea, lentil, grape, turf grass, Brassica or Arabidopsis thaliana homolog of the PPO of Amaranthus tuberculatus as set forth in SEQ ID NO:16.
7. A plant, progeny plant, plant cell, plant tissue or seed of the plant produced by the method of claim 5 , wherein said progeny plant, cell, tissue or seed contains within its genetic complement the mutant PPO gene that expresses a mutant PPO protein that is deleted at an amino acid position corresponding to Gly210 or Gly 211 in the Amaranthus tuberculatus PPO protein or at an analogous amino acid residue in a PPO homolog.
8. The plant, plant cell or plant tissue of claim 7 , which is or is from a dicotyledonous plant.
9. The plant, plant cell or plant tissue of claim 8 , which is or is from Arabidopsis , cotton, sunflower, cabbage, broccoli, cauliflower, Brussels sprout, canola, bean, pea, soybean, citrus, tomato, potato, sweet potato, plum, peach, apple, pear, cherry, grape, rose, tobacco, alfalfa, trefoil, sugar beet, blackberry, raspberry, blueberry, marionberry, loganberry, apricot, eggplant, pepper, pumpkin, squash, gourd, an ornamental plant, aspen, poplar, maple, oak, sunflower, rose or tobacco.
10. The plant, plant cell or plant tissue of claim 7 , which is or is from a monocotyledonous plant.
11. The plant, plant cell or plant tissue of claim 10 , which is or is from wheat, corn, rice, sorghum, oat, wild rice, barley, millet or turfgrass.
12. A method of controlling weeds comprising the step of planting a non-transgenic, genetically engineered, herbicide resistant plant, plant, progeny plant or seed containing within its genetic complement the mutant PPO gene that expresses a mutant PPO protein that is deleted at an amino acid position corresponding to Gly210 or Gly 211 in the Amaranthus tuberculatus PPO protein or at an analogous amino acid residue in a PPO homolog; and treating the plants with a herbicide to which resistance is conferred by said molecule, whereby growth of weeds is controlled.
13. A method of selecting a non-transgenic, genetically engineered, herbicide resistant plant cell or plant tissue containing within its genetic complement the mutant PPO gene that expresses a mutant PPO protein that is deleted at an amino acid position corresponding to Gly210 or Gly 211 in the Amaranthus tuberculatus PPO protein or at an analogous amino acid residue in a PPO homolog, said method comprising the steps of growing said plant cell or plant tissue on a medium comprising an amount of an inhibitor of protoporphoryinogen oxidase sufficient to prevent growth of a corresponding plant cell which does not contain and express said nucleic acid molecule.