Claims for Patent: 7,728,191

Summary for Patent: 7,728,191

Title:	Nucleic acid for reducing protein content in rice seed
Abstract:	It is intended to provide a method of reducing the total amount of stored protein, to develop a technique required therefor, and to provide a plant and its seeds developed by the above method and a method of using such a plant and seeds. More specifically, a nucleic acid molecule containing a consecutive nucleic acid sequence of at least 15 in length which is complementary with a nucleic acid sequence encoding prolamin polypeptide or a nucleic acid sequence having a homology of at least about 70% to the complementary nucleic acid sequence having at least 15 nucleotide length. A method of reducing the expression dose of a protein in a seed of a plant which comprises: A) the step of providing the above-described nucleic acid molecule; B) the step of transferring the nucleic acid molecule into the cells of the plant; C) the step of re-differentiating the cells to construct a transgenic plant; and D) the step of obtaining seeds from the transgenic plant.
Inventor(s):	Kuroda; Masaharu (Niigata, JP)
Assignee:	Incorporated Administrative Agency National Agriculture and Bio-oriented Research Organization (Ibaraki, JP) N/A (N/A)
Application Number:	10/539,992
Patent Claims:	1. An isolated nucleic acid molecule having antisense or RNA interference activity comprising a promoter that functions in a rice plant operably linked to: (i) a nucleic acid sequence having at least 15 contiguous nucleotides complementary to a gene encoding a rice prolamin polypeptide, or (ii) a nucleic acid sequence having at least 70% homology to (i), wherein, when introduced into a rice cell expressing the prolamin polypeptide, the nucleic acid is effective for reducing the amount of expression of the prolamin polypeptide relative to a rice plant into which the nucleic acid was not introduced. 2. The nucleic acid molecule according to claim 1 comprising said nucleic acid sequence having at least 15 contiguous nucleotides complementary to a gene encoding a prolamin polypeptide. 3. The nucleic acid molecule according to claim 1, wherein the prolamin is of japonica rice. 4. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence having at least 15 contiguous nucleotides complementary to a gene encoding a prolamin polypeptide is at least 50 nucleotides in length. 5. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence having at least 15 contiguous nucleotides complementary to a gene encoding a prolamin polypeptide comprises a full length sequence encoding the prolamin polypeptide. 6. The nucleic acid molecule according to claim 1, wherein the nucleic acid sequence having at least 15 contiguous nucleotides complementary to a gene encoding a prolamin polypeptide is complementary to the sequence encoding the signal peptide of said prolamin. 7. The nucleic acid molecule according to claim 1, wherein the at least 15 contiguous nucleotides complementary to a gene encoding a prolamin polypeptide is a polynucleotide of 50 nucleotides or less. 8. The nucleic acid molecule according to claim 1, wherein the at least 15 contiguous nucleotides complementary to a gene encoding a prolamin polypeptide is a polynucleotide of 30 nucleotides or less. 9. The nucleic acid molecule according to claim 1, wherein the prolamin is a 13 kDa prolamin. 10. The nucleic acid molecule according to claim 1, wherein said nucleic acid sequence of at least 15 contiguous nucleotides is complementary to: (a) a polynucleotide having a nucleic acid sequence set forth in a SEQ ID NO selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45, or a fragment sequence thereof; (b) a polynucleotide encoding a polypeptide having an amino acid sequence set forth in a SEQ ID NO selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46, or a fragment sequence thereof; (c) a polynucleotide encoding a polypeptide variant having at least one mutation selected from the group consisting of one or more amino acid substitution, addition and deletion in an amino acid sequence set forth in a SEQ ID NO selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46, and having a biological activity; (d) a polynucleotide that is an allelic variant of a DNA consisting of a nucleic acid sequence set forth in a SEQ ID NO selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45; (e) a polynucleotide encoding a species homolog or an ortholog of a polypeptide consisting of an amino acid sequence set forth in a SEQ ID NO selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46; (f) a polynucleotide hybridizing to at least one polynucleotide of any of (a)-(e), and encoding a polypeptide having a biological activity; or (g) a polynucleotide consisting of a base sequence having at least 70% identity with at least one polynucleotide of (a)-(e) or a complementary sequence thereof, and encoding a polypeptide having a biological activity. 11. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule has RNA interference activity, and wherein said molecule further comprises a polynucleotide complementary to the nucleic acid sequence. 12. The nucleic acid molecule according to claim 9, further comprising a spacer sequence. 13. The nucleic acid molecule according to claim 12, wherein the spacer sequence comprises an intron sequence. 14. The nucleic acid molecule according to claim 12, wherein the spacer sequence is between the nucleic acid sequence and the polynucleotide. 15. A nucleic acid cassette comprising a nucleic acid sequence having antisense or RNA interference activity, comprising: (i) a nucleic acid sequence having at least 15 contiguous nucleotides complementary to a gene encoding a rice prolamin polypeptide, or (ii) a nucleic acid sequence having at least about 70% homology to (i), wherein, when introduced into a rice cell expressing the prolamin polypeptide, the nucleic acid cassette is effective for reducing the amount of expression of the prolamin polypeptide relative to a rice plant into which the nucleic acid cassette was not introduced. 16. The nucleic acid cassette according to claim 15, further comprising a promoter that functions in rice operably linked to a nucleic acid sequence encoding a foreign protein. 17. The nucleic acid cassette according to claim 15, wherein said cassette has RNA interference activity, and wherein said cassette further comprises a polynucleotide complementary to the nucleic acid sequence. 18. The nucleic acid cassette according to claim 17, further comprising a spacer sequence. 19. The nucleic acid cassette according to claim 18, wherein the spacer sequence comprises an intron sequence. 20. The nucleic acid cassette according to claim 18, wherein the spacer sequence is between the nucleic acid sequence and the polynucleotide. 21. The nucleic acid cassette according to claim 16 or claim 17, further comprising a polynucleotide encoding a signal sequence fused, in frame, to the nucleic acid sequence encoding a foreign protein. 22. The nucleic acid cassette according to claim 21, wherein the signal sequence is a signal sequence of a storage protein. 23. The nucleic acid sequence according to claim 21, wherein the signal sequence is a prolamin signal sequence. 24. The nucleic acid cassette according to claim 16, wherein the promoter sequence is operably linked to both the nucleic acid sequence encoding the foreign protein and the polynucleotide. 25. The nucleic acid cassette according to claim 16, wherein separate promoters are independently operably linked to the nucleic acid sequence encoding the foreign protein and the polynucleotide. 26. The nucleic acid cassette according to claim 25, wherein a first promoter sequence is operably linked to the nucleic acid sequence encoding the foreign protein, and a second promoter sequence is operably linked to the polynucleotide, and the first and second promoter sequences are not the same. 27. The nucleic acid cassette according to claim 26, wherein the second promoter sequence promotes expression in a high level in seeds. 28. The nucleic acid cassette according to claim 26, wherein the second promoter sequence is derived from a storage protein promoter. 29. The nucleic acid cassette according to claim 26 wherein the second promoter sequence is derived from a promoter selected from the group consisting of a polyubiquitin promoter, 26 kD globulin promoter, glutelin A promoter, glutelin B promoter, 16 kD prolamin promoter, 13 kD prolamin promoter and 10 kD prolamin promoter. 30. The nucleic acid cassette according to claim 26 wherein the first promoter sequence is derived from a storage protein promoter. 31. The nucleic acid cassette according to claim 26, wherein the first promoter sequence is a promoter sequence naturally associated with the polynucleotide. 32. The nucleic acid cassette according to claim 26 wherein the first promoter sequence is derived from a promoter selected from the group consisting of 26 kD globulin promoter, glutelin A promoter, glutelin B promoter, 16 kD prolamin promoter, 13 kD prolamin promoter and 10 kD prolamin promoter. 33. The nucleic acid cassette according to claim 26, wherein the first promoter sequence is a prolamin promoter. 34. The nucleic acid cassette according to claim 26, wherein the first promoter sequence is derived from a prolamin promoter, and the second promoter sequence is derived from a promoter other than the prolamin promoter. 35. The nucleic acid cassette according to claim 16, comprising a polynucleotide encoding a signal sequence in frame between the nucleic acid encoding the foreign protein and the promoter sequence. 36. The nucleic acid cassette according to claim 17 further comprising a terminator sequence. 37. The nucleic acid cassette according to claim 36, wherein the terminator sequence is a terminator sequence of 10 kD prolamin. 38. The nucleic acid cassette according to claim 17, further comprising a nucleic acid encoding a foreign protein located upstream of both the polynucleotide and the nucleic acid complementary to said polynucleotide. 39. The nucleic acid cassette according to claim 38 comprising a spacer sequence between the polynucleotide and the nucleic acid complementary to said polynucleotide. 40. The nucleic acid cassette according to claim 38 comprising an intron sequence between the polynucleotide and the nucleic acid complementary to said polynucleotide. 41. A method for producing a transgenic rice plant comprising the steps of: A) providing the nucleic acid cassette according to claim 15; B) transforming a rice plant with the nucleic acid cassette; and C) selecting a transformed rice plant having a reduced amount of protein in the seeds compared to an untransformed rice plant. 42. A vector comprising the nucleic acid molecule according to claim 1. 43. The vector according to claim 42, wherein the promoter is a storage protein promoter. 44. The vector according to claim 42 wherein the promoter is a prolamin promoter. 45. The vector according to claim 42, further comprising a terminator. 46. The vector according to claim 42, further comprising a sequence encoding a selectable marker. 47. The vector according to claim 42, further comprising a sequence encoding a foreign protein. 48. A rice plant cell comprising the nucleic acid molecule according to claim 1. 49. The plant cell according to claim 48, further comprising a nucleic acid molecule encoding a foreign protein. 50. The rice plant cell according to claim 48 wherein the plant cell is from the same rice variety from which the prolamin is derived. 51. The rice plant cell according to claim 48, wherein the cell is of a japonica rice and the prolamin is from a japonica rice. 52. The rice plant cell according to claim 48, wherein the cell is homozygous for the nucleic acid molecule. 53. A plant tissue comprising the plant cell according to claim 48. 54. A starch preparation produced from the rice plant cell according to claim 48, wherein said starch preparation comprises said nucleic acid molecule. 55. A composition comprising a plant tissue comprising the plant cell according to claim 49, wherein said plant cell comprises said foreign protein. 56. A method for reducing the amount of protein in a seed of a rice plant, comprising the steps of: A) introducing the nucleic acid molecule of claim 1 into a rice plant cell; B) redifferentiating the cell to produce a transgenic rice plant; and C) obtaining a seed from the transgenic rice plant. 57. The method according to claim 56, wherein the step of introducing is performed by Agrobacterium-mediated transformation. 58. The method according to claim 56, further comprising the step of D) selecting a plant cell with the nucleic acid introduced therein. 59. The method according to claim 58, wherein the step of selecting is performed by determining resistance against an antibiotic. 60. A method for expressing a foreign protein in a rice plant seed, comprising the steps of: providing the nucleic acid molecule according to claim 1; providing a nucleic acid encoding the foreign protein; introducing the nucleic acid molecule according to claim 1 and the nucleic acid encoding the foreign protein into a cell of the rice plant; redifferentiating the cell to produce a transgenic rice plant; and obtaining a seed from the transgenic rice plant. 61. The method according to claim 60, wherein the step of introducing is performed by Agrobacterium-mediated transformation. 62. The method according to claim 60, further comprising the step of selecting a plant cell with the nucleic acid molecule introduced. 63. The method according to claim 62, wherein the step of selecting is carried out by determining resistance of the plant cell against an antibiotic. 64. The method according to claim 60, further comprising the step of separating the foreign protein from the seed.

Details for Patent 7,728,191

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2022-12-20
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2022-12-20
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2022-12-20