Polynucleotide encoding a maize herbicide resistance gene and methods for use

Abstract

This invention relates to polynucleotide sequences encoding a gene that can confer resistance to at least one herbicide. It further relates to plants and seeds of plants carrying chimeric genes comprising said polynucleotide sequences, which enhance or confer resistance to at least one herbicide, and methods of making said plants and seeds. The invention further presents sequences that can be used as molecular markers that in turn can be used to identify the region of interest in corn lines resulting from new crosses and to quickly and efficiently select the best lines for breeding strategies by avoiding sensitive lines.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1( a-e) is a multiple sequence alignment of the polypeptide sequence of the embodiments (SEQ ID NO: 2) comparing it to other known Cytochrome P450 polypeptides (SEQ ID NOs: 3-13). FIG. 1d also indicates the position of the most commonly conserved domain of the cytochrome P450 family (SEQ ID NO: 14). Identical residues in the alignment are indicated in upper case letters.

FIG. 2( a-b) is a multiple sequence alignment of the polypeptide sequences of several sensitive and resistant corn lines showing the commonly conserved domain of the cytochrome P450 family (SEQ ID NO: 14) as well as variations among the sequences.

Claims

1. An isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide capable of conferring resistance to at least one herbicide, wherein said herbicide is a member of a class of herbicides selected from the group consisting of: (i) the ALS-inhibiting class;(ii) the pigment synthesis-inhibiting class;(iii) the PPO-inhibiting class;(iv) the PS II-inhibiting class; and(v) the synthetic auxin class

2. An isolated polynucleotide of claim 1, wherein the polypeptide is capable of conferring resistance to at least two herbicides, wherein each herbicide is a member of a different class of herbicides selected from the group consisting of: (a) the ALS-inhibiting class;(b) the pigment synthesis-inhibiting class;(c) the PPO-inhibiting class;(d) the PS II-inhibiting class; and(e) the synthetic auxin class.

3. An isolated polynucleotide of claim 1, wherein the polypeptide is capable of conferring resistance to at least three herbicides, wherein each herbicide is a member of a different class of herbicides selected from the group consisting of: (a) the ALS-inhibiting class;(b) the pigment synthesis-inhibiting class;(c) the PPO-inhibiting class;(d) the PS II-inhibiting class; and(e) the synthetic auxin class.

4. An isolated polynucleotide of claim 1, wherein the polypeptide is capable of conferring resistance to at least four herbicides, wherein each herbicide is a member of a different class of herbicides selected from the group consisting of: (a) the ALS-inhibiting class;(b) the pigment synthesis inhibiting class;(c) the PPO-inhibiting class;(d) the PS II-inhibiting class; and(e) the synthetic auxin class.

5. An isolated polynucleotide of claim 1, wherein the polypeptide is capable of conferring resistance to at least five herbicides, wherein each herbicide is a member of a different class of herbicides selected from the group consisting of: (a) the ALS-inhibiting class;(b) the pigment synthesis-inhibiting class;(c) the PPO-inhibiting class;(d) the PS II-inhibiting class; and(e) the synthetic auxin class.

6. The polynucleotide of claim 1, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO: 1 have at least 90% identity based on the Needleman-Wunsch alignment algorithm.

7. The polynucleotide of claim 1, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO: 1 have at least 95% identity based on the Needleman-Wunsch alignment algorithm.

8. The polynucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO: 1.

9. A vector comprising the polynucleotide of claim 1.

10. A recombinant DNA construct comprising the polynucleotide of claim 1 operably linked to at least one regulatory sequence.

11. A method for transforming a cell, comprising transforming a cell with the polynucleotide of claim 1.

12. A plant cell comprising the recombinant DNA construct of claim 10.

13. A method for producing a plant comprising transforming a plant cell with the recombinant DNA construct of claim 10 and regenerating a plant from the transformed plant cell.

14. A plant comprising the recombinant DNA construct of claim 10.

15. A seed comprising the recombinant DNA construct of claim 10.

16. The plant of claim 14, wherein said plant is a monocot.

17. The plant of claim 16, wherein said dicot is selected from the group consisting of maize, wheat, barley, oats, switchgrass, sorghum, and rice.

18. The plant of claim 14, wherein said plant is a dicot.

19. The plant of claim 18, wherein said dicot is selected from the group consisting of soybean, canola, potato, cotton, and sunflower.

20. The plant of claim 14, wherein said plant further comprises a second herbicide resistance gene.

21. The plant of claim 14, wherein said plant further comprises a gene encoding a polypeptide with glyphosate N-acetyltransferase activity.

22. The plant of claim 21, wherein said plant further comprises a second herbicide resistance gene encoding a polypeptide conferring tolerance to ALS inhibitors.

23. The plant of claim 14, wherein said plant further comprises a gene encoding an insecticidal polypeptide.

24. A plant with enhanced tolerance to at least one herbicide, comprising the recombinant DNA construct of claim 10, wherein said plant further comprises a second herbicide resistance gene providing a level of tolerance to a herbicide selected from a class of herbicides selected from the group consisting of: (a) the ALS-inhibiting class;(b) the pigment synthesis-inhibiting class;(c) the PPO-inhibiting class;(d) the PS II-inhibiting class; and(e) the synthetic auxin class;

25. A method of conferring or enhancing resistance to at least one herbicide, wherein said herbicide is selected from a class of herbicides selected from the group consisting of: (a) the ALS-inhibiting class;(b) the pigment synthesis-inhibiting class;(c) the PPO-inhibiting class;(d) the PS II-inhibiting class; and(e) the synthetic auxin class;

26. A method of conferring or enhancing resistance to at least two herbicides, wherein each herbicide is selected from a class of herbicides selected from the group consisting of: (a) the ALS-inhibiting class;(b) the pigment synthesis-inhibiting class;(c) the PPO-inhibiting class;(d) the PS II-inhibiting class; and(e) the synthetic auxin class;

27. A method of conferring or enhancing resistance to at least three herbicides, wherein said each herbicide is selected from a class of herbicides selected from the group consisting of: (a) the ALS-inhibiting class;(b) the pigment synthesis-inhibiting class;(c) the PPO-inhibiting class;(d) the PS II-inhibiting class; and(e) the synthetic auxin class;

28. A method of conferring or enhancing resistance to at least four herbicides, wherein each herbicide is selected from a class of herbicides selected from the group consisting of: (a) the ALS-inhibiting class;(b) the pigment synthesis-inhibiting class;(c) the PPO-inhibiting class;(d) the PS II-inhibiting class; and(e) the synthetic auxin class;

29. A method of conferring or enhancing resistance to at least five herbicides, wherein each herbicide is selected from a class of herbicides selected from the group consisting of: (a) the ALS-inhibiting class;(b) the pigment synthesis-inhibiting class;(c) the PPO-inhibiting class;(d) the PS II-inhibiting class; and(e) the synthetic auxin class;

30. A method of altering the level of expression of a protein capable of conferring resistance to at least one herbicide in a plant cell comprising: (a) transforming a plant cell with the recombinant DNA construct of claim 10; and(b) growing the transformed plant cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of a protein capable of conferring resistance to the at least one herbicide in the transformed host;

31. A method of altering the level of expression of a protein capable of conferring resistance to at least two herbicides in a plant cell comprising: (a) transforming a plant cell with the recombinant DNA construct of claim 10; and(b) growing the transformed plant cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of a protein capable of conferring resistance to the at least two herbicides in the transformed host;

32. A method of altering the level of expression of a protein capable of conferring resistance to at least three herbicides in a plant cell comprising: (a) transforming a plant cell with the recombinant DNA construct of claim 10; and(b) growing the transformed plant cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of a protein capable of conferring resistance to the at least three herbicides in the transformed host;wherein each of the at least three herbicides is selected from a class of herbicides selected from the group consisting of:(i) the ALS-inhibiting class;(ii) the pigment synthesis-inhibiting class;(iii) the PPO-inhibiting class;(iv) the PS II-inhibiting class; and(v) the synthetic auxin class.

33. A method of altering the level of expression of a protein capable of conferring resistance to at least four herbicides in a plant cell comprising: (a) transforming a plant cell with the recombinant DNA construct of claim 10; and(b) growing the transformed plant cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of a protein capable of conferring resistance to the at least four herbicides in the transformed host;wherein each of the at least four herbicides is selected from a class of herbicides selected from the group consisting of:(i) the ALS-inhibiting class;(ii) the pigment synthesis-inhibiting class;(iii) the PPO-inhibiting class;(iv) the PS II-inhibiting class; and(v) the synthetic auxin class.

34. A method of altering the level of expression of a protein capable of conferring resistance to at least five herbicides in a plant cell comprising: (a) transforming a plant cell with the recombinant DNA construct of claim 10; and(b) growing the transformed plant cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of a protein capable of conferring resistance to the at least five herbicides in the transformed host;

35. The method of any one of claims 25-34 wherein the herbicide is selected from the ALS-inhibiting class of herbicides and is selected from the group consisting of: (a) nicosulfuron;(b) rimsulfuron;(c) primisulfuron;(d) imazethapyr;(e) chlorsulfuron;(f) chlorimuron ethyl;(g) triasulfuron;(h) flumetsulam; and(i) imazaquin.

36. The method of any one of claims 25-34 wherein the herbicide is selected from the pigment synthesis-inhibiting class of herbicides and is selected from the group consisting of: (a) isoxaflutole;(b) topramezone;(c) sulcatrione; and(d) tembotrione.

37. The method of any one of claims 25-34 wherein the herbicide is selected from the PPO-inhibiting class of herbicides and is selected from the group consisting of: (a) acifluorfen;(b) flumioxan; and(c) sulfentrazone.

38. The method of any one of claims 25-34 wherein the herbicide is selected from the PS II-inhibiting class of herbicides and is selected from the group consisting of: (a) diuron;(b) linuron;(c) bentazon; and(d) chlorotoluron.

39. The method of any one of claims 25-34 wherein the herbicide is dicamba.

40. A method of determining the presence of the polynucleotide of claim 1 in a plant, comprising at least one of: (a) isolating nucleic acid molecules from said plant and determining if an Nsf1 gene is present by attempting to amplify sequences homologous to the polynucleotide of claim 1, or(b) isolating nucleic acid molecules from said plant and performing a Southern or northern hybridization, or (c) isolating proteins from said plant and performing a western blot using antibodies to the NSF1 protein, or(d) isolating proteins from said plant and performing an ELISA assay using antibodies to the NSF1 protein, thereby determining the presence of the polynucleotide of claim 1 in said plant.

41. A method of determining the presence of the Nsf1 locus in a plant, comprising at least one of: (a) isolating nucleic acid molecules from said plant and determining if an Nsf1 gene is present by attempting to amplify sequences homologous to the polynucleotide of claim 1, or(b) isolating nucleic acid molecules from said plant and performing a Southern or northern hybridization, or (c) isolating proteins from said plant and performing a western blot using antibodies to the NSF1 protein, or(d) isolating proteins from said plant and performing an ELISA assay using antibodies to the NSF1 protein, thereby determining the presence of the Nsf1 locus in said plant.

42. A soybean plant comprising the polynucleotide of claim 1, wherein said soybean plant also exhibits soybean cyst nematode resistance, wherein said polynucleotide has been incorporated through transformation or plant breeding techniques, and wherein said soybean plant has been bred from germplasm selected from the group consisting of: (a) Peking;(b) P188788;(c) P189772;(d) P190763;(e) P1209332;(f) P1404189A;(g) P1437654;(h) P1438489B;(i) P1467312;(j) P1468916;(k) Hartwig;(l) J87-233; and(m) progeny derived from sources (a) through (l).