Gene silencing

• US 20050138689A1
• Filed: 10/12/2004
• Published: 06/23/2005
• Est. Priority Date: 10/09/2003
• Status: Abandoned Application

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Aukerman, Milo J.
• Application Number:
  US10/963,394
• Publication Number:
  US 20050138689A1
• Time in Patent Office: Days
• US Class Current: 800/286
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/827 Flower development or morphology, e.g. flowering promoting factor [FPF]

Abstract

The invention provides methods and compositions useful in target sequence suppression and target sequence validation. The invention provides polynucleotide constructs useful for gene silencing, as well as cells, plants and seeds comprising the polynucleotides. The invention also provides a method for using microRNA to silence a target sequence.

Claims

1. A method of inhibiting expression of a target sequence in a cell comprising:

(a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide, the polynucleotide comprising in the following order;

(i) at least about 20 contiguous nucleotides in the region from nucleotides 1-38 of SEQ ID NO;

3,

(ii) a first oligonucleotide of 10 to about 50 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide,

(iii) at least about 20 contiguous nucleotides in the region from nucleotides 60-106 of SEQ ID NO;

3,

(iv) the second oligonucleotide of about 10 to about 50 contiguous nucleotides, wherein the second oligonucleotide encodes a mRNA, and wherein the second oligonucleotide is substantially complementary to the target sequence, and

(v) at least about 20 contiguous nucleotides in the region from nucleotides 128-159 of SEQ ID NO;

3;

wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin, and

(b) expressing the nucleic acid construct for a time sufficient to produce the mRNA, wherein the mRNA inhibits expression of the target sequence.

2. The method of claim 1, wherein the nucleic acid construct further comprises a promoter operably linked to the polynucleotide

3. The method of claim 1, wherein the cell is a plant cell.

4. A cell comprising the nucleic acid construct of claim 1.

5. The cell of claim 4, wherein the cell is a plant cell.

6. The method of claim 1, wherein target sequence expression is inhibited by at least 10%.

7. The method of claim 1, wherein inhibition of the target sequence generates a loss-of-function phenotype.

8. The method of claim 2, wherein the promoter is a pathogen-inducible promoter and inhibition of the target sequence confers resistance to a pathogen.

26. A cell produced by the method of claim 1.

27. The cell of claim 26, wherein the cell is a plant cell.

28. The cell of claim 27, wherein the plant cell is from a monocot or a dicot.

29. The cell of claim 28, wherein the cell is selected from the group consisting of corn, wheat, rice, barley, oats, sorghum, millet, sunflower, safflower, cotton, soy, canola, alfalfa, Arabidopsis, and tobacco.

30. The cell of claim 29, wherein the cell is from Arabidopsis.

31. The cell of claim 29, wherein the cell is from corn.

50. The method of claim 3, the method further comprising producing a transformed plant, wherein the plant comprises the nucleic acid construct which encodes the mRNA.

51. A plant produced by the method of claim 50.

55. A plant produced by the method of claim 50.

9. An isolated polynucleotide comprising in the following order at least 20 contiguous nucleotides in the region from nucleotides 1-38 of SEQ ID NO:

3, a first oligonucleotide of about 10 to about 50 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide, at least about 20 contiguous nucleotides in the region from nucleotides 60-106 of SEQ ID NO;

3, a second oligonucleotide of about 10 to about 50 contiguous nucleotides, wherein the second oligonucleotide encodes a mRNA, and the second oligonucleotide is substantially complementary to the target sequence, and at least about 20 contiguous nucleotides in the region from nucleotides 128-159 of SEQ ID NO;

3, wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin.

10. The isolated polynucleotide of claim 9, further comprising an operably linked promoter.

11. A cell comprising the isolated polynucleotide of claim 9.

12. The cell of claim 11, wherein the cell is a plant cell.

13. A transgenic plant comprising the isolated polynucleotide of claim 9.

14. A transgenic seed comprising the isolated polynucleotide of claim 9.

15. The isolated polynucleotide of claim 10, wherein the promoter is a pathogen-inducible promoter.

16. The isolated polynucleotide of claim 9, wherein the polynucleotide suppresses expression of a target sequence.

17. A method of inhibiting expression of a target sequence in a cell comprising:

(a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide, wherein the polynucleotide comprises in the following order;

(i) nucleotides 1-38 of SEQ ID NO;

3,

(ii) a first oligonucleotide of 21 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide,

(iii) nucleotides 60-106 of SEQ ID NO;

3,

(iv) a second oligonucleotide of 21 contiguous nucleotides, wherein the second oligonucleotide encodes a mRNA, and wherein the second oligonucleotide is substantially complementary to the target sequence, and

(v) nucleotides 128-159 of SEQ ID NO;

3;

wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin, and

(b) expressing the nucleic acid construct for a time sufficient to produce the mRNA, wherein the mRNA inhibits expression of the target sequence.

18. An isolated polynucleotide comprising in the following order nucleotides 1-38 of SEQ ID NO:

3, a first oligonucleotide of 21 contiguous nucleotides, wherein the first oligonucleotide is substantially complementary to a second oligonucleotide, nucleotides 60-106 of SEQ ID NO;

3, a second oligonucleotide of 21 contiguous nucleotides, wherein the second oligonucleotide encodes a mRNA, and the second oligonucleotide is substantially complementary to the target sequence, and nucleotides 128-159 of SEQ ID NO;

3, wherein the polynucleotide encodes an RNA precursor capable of forming a hairpin.

19. A method of inhibiting expression of a target sequence in a cell comprising:

(a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified plant mRNA precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide encodes a mRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin; and

(b) expressing the nucleic acid construct for a time sufficient to produce the mRNA, wherein the mRNA inhibits expression of the target sequence.

32. A cell produced by the method of claim 19.

33. The cell of claim 32, wherein the cell is a plant cell.

34. The cell of claim 33, wherein the plant cell is from a monocot or a dicot.

35. The cell of claim 34, wherein the cell is selected from the group consisting of corn, wheat, rice, barley, oats, sorghum, millet, sunflower, safflower, cotton, soy, canola, alfalfa, Arabidopsis, and tobacco.

36. The cell of claim 35, wherein the cell is from Arabidopsis.

37. The cell of claim 35, wherein the cell is from corn.

52. The method of claim 19, wherein the cell is a plant cell, the method further comprising producing a transformed plant, wherein the plant comprises the nucleic acid construct which encodes the mRNA.

53. A plant produced by the method of claim 52.

20. A method of inhibiting expression of a target sequence in a cell comprising:

(a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified plant miR172 mRNA precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide encodes a mRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin; and

(b) expressing the nucleic acid construct for a time sufficient to produce the mRNA, wherein the mRNA inhibits expression of the target sequence.

21. The method of claim 20, wherein the plant miR172 mRNA precursor is from a dicot or a monocot.

22. The method of claim 20, wherein the plant miR172 mRNA precursor is from Arabidopsis.

23. The method of claim 20, wherein the plant miR172 mRNA precursor comprises SEQ ID NO:

3.

38. A cell produced by the method of claim 20.

39. The cell of claim 38, wherein the cell is a plant cell.

40. The cell of claim 39, wherein the plant cell is from a monocot or a dicot.

41. The cell of claim 40, wherein the cell is selected from the group consisting of corn, wheat, rice, barley, oats, sorghum, millet, sunflower, safflower, cotton, soy, canola, alfalfa, Arabidopsis, and tobacco.

42. The cell of claim 41, wherein the cell is from Arabidopsis.

43. The cell of claim 41, wherein the cell is from corn.

54. The method of claim 20, wherein the cell is a plant cell, the method further comprising producing a transformed plant, wherein the plant comprises the nucleic acid construct which encodes the mRNA.

24. A method of inhibiting expression of a target sequence in a cell comprising:

(a) introducing into the cell a nucleic acid construct comprising a promoter operably linked to a polynucleotide encoding a modified Arabidopsis miR172 mRNA precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide encodes a mRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin; and

(b) expressing the nucleic acid construct for a time sufficient to produce the mRNA, wherein the mRNA inhibits expression of the target sequence.

25. The method of claim 24, wherein the Arabidopsis miR172 mRNA precursor comprises SEQ ID NO:

3.

44. A cell produced by the method of claim 24.

45. The cell of claim 44, wherein the cell is a plant cell.

46. The cell of claim 45, wherein the plant cell is from a monocot or a dicot.

47. The cell of claim 46, wherein the cell is selected from the group consisting of corn, wheat, rice, barley, oats, sorghum, millet, sunflower, safflower, cotton, soy, canola, alfalfa, Arabidopsis, and tobacco.

48. The cell of claim 47, wherein the cell is from Arabidopsis.

49. The cell of claim 47, wherein the cell is from corn.

56. The method of claim 24, wherein the cell is a plant cell, the method further comprising producing a transformed plant, wherein the plant comprises the nucleic acid construct which encodes the mRNA.

57. A plant produced by the method of claim 56.

58. The plant of claim 57, wherein the plant is a monocot or a dicot.

59. The plant of claim 58, wherein the plant is selected from the group consisting of corn, wheat, rice, barley, oats, sorghum, millet, sunflower, safflower, cotton, soy, canola, alfalfa, Arabidopsis, and tobacco.

60. The plant of claim 59, wherein the plant is Arabidopsis.

61. The plant of claim 59, wherein the plant is corn.

62. An isolated polynucleotide comprising a polynucleotide which encodes a modified plant mRNA precursor, the modified precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide comprises a mRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin.

63. A cell comprising the isolated polynucleotide of claim 62.

64. The cell of claim 63, wherein the cell is a plant cell.

74. The isolated polynucleotide of claim 62, wherein the first and the second oligonucleotides are heterologous to the precursor.

65. An isolated polynucleotide comprising a polynucleotide encoding a modified plant miR172 mRNA precursor, the modified precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide comprises a mRNA substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin.

66. A cell comprising the isolated polynucleotide of claim 65.

67. The cell of claim 66, wherein the cell is a plant cell.

75. The isolated polynucleotide of claim 65, wherein the first and the second oligonucleotides are heterologous to the precursor.

68. An isolated polynucleotide comprising polynucleotide encoding a modified Arabidopsis miR172 mRNA precursor, the modified precursor comprising a first and a second oligonucleotide, wherein at least one of the first or the second oligonucleotides is heterologous to the precursor, wherein the first oligonucleotide is substantially complementary to the second oligonucleotide, and the second oligonucleotide comprises a mRNA which is substantially complementary to the target sequence, wherein the precursor is capable of forming a hairpin.

69. A cell comprising the isolated polynucleotide of claim 68.

70. The cell of claim 69, wherein the cell is a plant cell.

71. The cell of claim 70, wherein the plant cell is from a dicot or a monocot.

72. The cell of claim 71, wherein the cell is from Arabidopsis.

73. The cell of claim 71, wherein the cell is from corn.

76. The isolated polynucleotide of claim 68, wherein the first and the second oligonucleotides are heterologous to the precursor.