Claims
- 1. A transgenic plant having an altered trait as compared to a wild-type or untransformed plant, wherein said altered trait is selected from the group consisting of:
(a) altered levels of at least one cell protectant in cells of said transgenic plant; and (b) altered biomass; wherein said transgenic plant is transformed with a recombinant polynucleotide comprising a nucleotide sequence that encodes a CBF, and said CBF comprises an AP2 domain; wherein the altered levels of at least one cell protectant is selected from the group consisting of
(i) increasing; and (ii) decreasing levels; and wherein the altered biomass is selected from the group consisting of:
(i) increasing; and (ii) decreasing biomass.
- 2. The transgenic plant of claim 1, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof.
- 3. The transgenic plant of claim 1, wherein said CBF is selected from the group consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, and fragments thereof.
- 4. The transgenic plant of claim 1, wherein said nucleotide sequence specifically hybridizes under highly stringent conditions to SEQ ID NO: 1 or its complement under the conditions of 6×SSC and 65° C.
- 5. The transgenic plant of claim 1, wherein expression of said CBF modifies the level of said at least one cell protectant in said cells of said transgenic plant.
- 6. The transgenic plant of claim 1, wherein said transgenic plant is a crop plant selected from the group consisting of Brassica juncea, Brassica napus, Brassica oleracea, Brassica rapa, Brassica rapa L, Brassica napus L, Glycine max, Raphanus sativus, Zea mays, Triticum, Oryza sativa, Secale cereale, Sorghum bicolor, Sorghum vulgare, and Hordeum vulgare.
- 7. The transgenic plant of claim 1, wherein said transgenic plant has increased tolerance to an environmental stress.
- 8. The transgenic plant of claim 7, wherein said environmental stress is selected from the group consisting of drought, cold, freezing, and high salt.
- 9. A transgenic plant having an altered trait as compared to a wild-type or untransformed plant, wherein said altered trait is selected from the group consisting of:
(a) altered levels of at least one cell protectant in cells of said transgenic plant; and (b) altered biomass; wherein said transgenic plant is transformed with a recombinant polynucleotide comprising a nucleotide sequence that encodes a CBF, and said CBF comprises a sequence selected from the group consisting of:
(a) an AP2 domain comprising consecutive amino acid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein amino acids 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102, and 104-106 of SEQ ID NO:2 are conserved in the AP2 domain of the CBF; and wherein the alteration in levels of the at least one cell protectant is selected from the group consisting of (i) increasing; and (ii) decreasing levels; and wherein the altered biomass is selected from the group consisting of (i) increasing; and (ii) decreasing biomass.
- 10. The transgenic plant of claim 9, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof.
- 11. The transgenic plant of claim 9, wherein said CBF is selected from the group consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, and fragments thereof
- 12. A transgenic rapeseed plant having altered biomass as compared to a wild-type or untransformed plant, wherein said transgenic rapeseed plant is transformed with a recombinant polynucleotide comprising a nucleotide sequence that encodes a CBF comprising an AP2 domain; and
wherein the altered biomass is selected from the group consisting of (i) increasing; and (ii) decreasing biomass.
- 13. The transgenic rapeseed plant of claim 12, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof.
- 14. The transgenic rapeseed plant of claim 12, wherein said CBF is selected from the group consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, and fragments thereof
- 15. The transgenic rapeseed plant of claim 12, wherein said nucleotide sequence specifically hybridizes under highly stringent conditions to SEQ ID NO: 1 or its complement under the conditions of 6×SSC and 65° C.
- 16. The transgenic rapeseed plant of claim 12, wherein said nucleotide sequence further comprises a regulatory region operably linked to the sequence encoding the CBF.
- 17. The transgenic rapeseed plant of claim 16, wherein said regulatory region is a constitutive promoter, an inducible promoter, a tissue specific promoter, or a developmental stage specific promoter.
- 18. A transgenic rapeseed plant having altered biomass, said transgenic rapeseed plant comprising a recombinant polynucleotide comprising a nucleotide sequence encoding a CBF comprises a sequence selected from the group consisting of:
(a) an AP2 domain comprising consecutive amino acid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102, and 104-106 of SEQ ID NO:2 are conserved in the AP2 domain of the CBF; and wherein said biomass is altered compared to that of a plant that has not been transformed with said recombinant polynucleotide; and wherein the altered biomass is selected from the group consisting of (i) increasing; and (ii) decreasing biomass.
- 19. A transgenic rapeseed plant having increased environmental stress tolerance, said transgenic rapeseed plant comprising a recombinant polynucleotide comprising a nucleotide sequence encoding a CBF comprising an AP2 domain.
- 20. The transgenic rapeseed plant of claim 19, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof.
- 21. The transgenic rapeseed plant of claim 19, wherein said CBF is selected from the group consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, and fragments thereof.
- 22. The transgenic rapeseed plant of claim 19, wherein said nucleotide sequence specifically hybridizes under highly stringent conditions to SEQ ID NO: 1 or its complement under the conditions of 6×SSC and 65° C.
- 23. The transgenic rapeseed plant of claim 19, wherein said CBF comprises a sequence selected from the group consisting of:
(a) an AP2 domain comprising consecutive amino acid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102, and 104-106 of SEQ ID NO:2 are conserved in the AP2 domain of the CBF.
- 24. The transgenic rapeseed plant of claim 19, wherein said recombinant polynucleotide further comprises a regulatory region operably linked to the sequence encoding the CBF.
- 25. The transgenic rapeseed plant of claim 24, wherein said regulatory region is a constitutive promoter, an inducible promoter, a tissue specific promoter or a developmental stage specific promoter.
- 26. The transgenic rapeseed plant of claim 19, wherein said environmental stress is selected from the group consisting of dehydration stress, cold stress, and freezing stress.
- 27. A cold-inducible promoter comprising a nucleotide sequence selected from the group consisting of nucleotides 1-1695 of SEQ ID NO: 129 and nucleotides 1-1952 of SEQ ID NO: 130.
- 28. The cold-inducible promoter of claim 27, wherein said nucleotide sequence is selected from the group consisting of nucleotides 1470-1520 of SEQ ID NO: 129 and nucleotides 460-520 of SEQ ID NO: 130.
- 29. A method for modifying the levels of a cell protectant in a cell, said method comprising:
(a) transforming the cell with a recombinant polynucleotide comprising a nucleotide sequence encoding a CBF; and (b) expressing said CBF in the transformed cell; wherein said transformed cell has altered cell protectant levels compared with a cell that has not been transformed with said recombinant polynucleotide.
- 30. The method of claim 29, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof.
- 31. The method of claim 29, wherein said CBF is selected from the group consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, and fragments thereof
- 32. The method of claim 29, wherein said nucleotide sequence specifically hybridizes under highly stringent conditions to SEQ ID NO: 1 or its complement under the conditions of 6×SSC and 65° C.
- 33. The method of claim 29, wherein said cell protectant is selected from the group consisting of proline, sucrose, and a fatty acid.
- 34. The method of claim 29, wherein said cell protectant is a fatty acid selected from the group consisting of 16:1, 16:2, or 18:0, and 18:1 fatty acids.
- 35. The method of claim 29, wherein said CBF binds to a member of a class of DNA regulatory sequences that includes a subsequence selected from the group consisting of CCGAA, CCGAT, CCGAC, CCGAG, CCGTA, CCGTT, CCGTC, CCGTG, CCGCA, CCGCT, CCGCG, CCGCC, CCGGA, CCGGT, CCGGC, CCGGG, AACCG, ATCCG, ACCCG, AGCCG, TACCG, TTCCG, TCCCG, TGCCG, CACCG, CTCCG, CGCCG, CCCCG, GACCG, GTCCG, GCCCG, GGCCG, ACCGA, ACCGT, ACCGC, ACCGG, TCCGA, TCCGT, TCCGC, TCCGG, CCCGA, CCCGT, CCCGC, CCCGG, GCCGA, GCCGT, GCCGC, and GCCGG.
- 36. The method of claim 29, wherein said CBF binds to a cold or dehydration transcription-regulating region comprising the sequence CCG.
- 37. The method of claim 29, wherein said CBF comprises a sequence selected from the group consisting of:
(a) an AP2 domain comprising consecutive amino acid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102, and 104-106 of SEQ ID NO:2 are conserved in the AP2 domain of the CBF.
- 38. The method of claim 29, wherein said CBF is CBF3.
- 39. The method of claim 29, further comprising cold-acclimating said transformed cell.
- 40. The method of claim 29, wherein said recombinant polynucleotide further comprises a regulatory region operably linked to the sequence encoding the CBF.
- 41. The method of claim 40, wherein said regulatory region is a constitutive promoter, an inducible promoter, a tissue specific promoter, or a developmental stage specific promoter.
- 42. The method of claim 29, wherein said CBF comprises an amino acid sequence homologous to a sequence selected from an amino acid sequence depicted in FIG. 19A, 19B, 19C, 19D, or 19E that binds to a DNA regulatory sequence that induces expression of an environmental stress tolerance gene and modifies cell protectant levels.
- 43. The method of claim 29, wherein the amino acid sequence comprises consecutive amino acid residues of Thr-Xaa(13)-Ala-Xaa(12)-Ser, wherein Xaa represents any amino acid residue.
- 44. The method of claim 29, wherein the amino acid sequence comprises consecutive amino acid residues of Asn-Xaa(12)-Thr-Xaa(13)-Ala-Leu-Arg-Xaa(8)-Ala-Xaa-Ser, wherein Xaa represents any amino acid residue.
- 45. The method of claim 29, wherein the amino acid sequence comprises consecutive amino acid residues of Gly-Val-Arg-Xaa-Arg-Tyr-Xaa(4-5)-Trp-Val-Xaa-Glu-Xaa-Arg-Glu-Xaa(6)-Arg-Glu-Xaa-Asn-Lys-Xaa(2)-Arg-Ile-Trp-Xaa-Gly-Thr-Phe-Xaa(5)-Ala-Ala-Xaa-Ala-Xaa-Asp-Xaa-Ala-Ala-Xaa(4)-Gly-Xaa(2)-Ala-Xaa-Leu-Asn, wherein Xaa represents any amino acid residue.
- 46. The method of claim 29, wherein the amino acid sequence comprises consecutive amino acid residues of Gly-Val-Arg-Xaa-Arg-Tyr-Xaa(4-5)-Trp-Val-Xaa-Glu-Xaa-Arg-Glu-Xaa(6)-Arg-Glu-Xaa-Asn-Lys-Xaa(2)-Arg-Ile-Trp-Xaa-Gly-Thr-Phe-Xaa-Thr-Xaa(3)-Ala-Ala-Xaa-Ala-Xaa-Asp-Xaa-Ala-Ala-Xaa-Ala-Xaa(2)-Gly-Xaa(2)-Ala-Xaa-Leu-Asn-Xaa(3)-Ser, wherein Xaa represents any amino acid residue.
- 47. The method of claim 29, wherein the amino acid sequence comprises consecutive amino acid residues of His-Pro-Xaa-Tyr-Gly-Val-Arg-Xaa-Arg-Tyr-Xaa(4-5)-Trp-Val-Xaa-Glu-Xaa-Arg-Glu-Xaa-Asn-Lys-Xaa(2)-Arg-Glu-Xaa-Asn-Lys-Xaa(2)-Arg-Ile-Trp-Xaa-Gly-Thr-Phe-Xaa-Thr-Xaa-Glu-Xaa-Ala-Ala-Arg-Ala-Asp-His-Asp-Val-Ala-Ala-Xaa-Ala-Leu-Arg-Gly-Xaa(2)-Ala-Xaa-Leu-Asn-Xaa-Ala-Asp-Ser, wherein Xaa represents any amino acid residue.
- 48. The method of claim 29, wherein said recombinant polynucleotide encodes a polypeptide that elevates cold-regulated gene levels in the absence of cold acclimation compared with cold-regulated gene levels in a plant lacking said recombinant polynucleotide.
- 49. A method for modifying the levels of a cell protectant in a plant cell, said method comprising:
(a) transforming the plant cell with a recombinant polynucleotide comprising a nucleotide sequence encoding a CBF; wherein said CBF is sufficiently homologous to a consensus sequence shown in depicted in FIG. 19A, 19B, 19C, 19D, or 19E that the CBF is capable of binding to a CCG regulatory sequence and that the nucleotide sequence that encodes the CBF can hybridize to SEQ ID NO: 1 under the conditions of 6×SSC and 65° C.; and (b) expressing said CBF in the transformed plant cell; wherein said transformed plant cell has altered cell protectant levels compared with a plant cell that has not been transformed with said recombinant polynucleotide.
- 50. A method for modifying proline, sucrose or fatty acid levels in a plant cell, said method comprising:
a) transforming the plant cell with a recombinant polynucleotide comprising a nucleotide sequence encoding a CBF; and (b) expressing said CBF in the transformed cell; whereby expression of said CBF modifies proline levels in the plant cell.
- 51. The method of claim 50, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof.
- 52. The method of claim 50, wherein said CBF is selected from the group consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, and fragments thereof.
- 53. The method of claim 50, wherein said nucleotide sequence specifically hybridizes under highly stringent conditions to SEQ ID NO: 1 or its complement under the conditions of 6×SSC and 65° C.
- 54. The method of claim 50, wherein said CBF comprises a sequence selected from the group consisting of:
(a) an AP2 domain comprising consecutive amino acid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and 104-106 of SEQ ID NO:2 are conserved in the AP2 domain of the CBF.
- 55. The method of claim 50, wherein said CBF is CBF3.
- 56. The method of claim 50, wherein said recombinant polynucleotide further comprises a regulatory region operably linked to the sequence encoding the CBF.
- 57. The method of claim 56, wherein said regulatory region is a constitutive promoter, an inducible promoter, a tissue specific promoter, or a developmental stage specific promoter.
- 58. A method for improving the tolerance of a plant cell to an environmental stress, said method comprising
(a) transforming the plant cell with a recombinant polynucleotide comprising a nucleotide sequence encoding a CBF; wherein said CBF is sufficiently homologous to a consensus sequence shown in depicted in FIG. 19A, 19B, 19C, 19D, or 19E that the CBF is capable of binding to a CCG regulatory sequence and that the nucleotide sequence that encodes the CBF can hybridize to SEQ ID NO: 1 under highly stringent conditions of 6×SSC and 65° C.; and (b) expressing said CBF in the transformed plant cell; whereby expression of said CBF increases cell protectant levels at least 1.5 fold in the transformed plant cell compared with cell protectant levels in an untransformed plant cell and the increased cell protectant levels in the transformed plant cell improve the tolerance of the transformed plant cell to the environmental stress, and wherein the environmental stress is selected from the group consisting of:
cold, freezing, dehydration, and drought; and wherein the improved environmental stress tolerance is selected from the group consisting of:
a) a decrease in the extent of a plant's injury; b) a decrease in the extent of a cell's injury; c) a decrease in the extent of a plant's growth inhibition; d) a decrease in the extent of a cell's growth inhibition; e) an increase in survival rate after exposure to cold temperatures; f) an increase in survival rate after exposure to freezing temperatures; g) an increase in the extent of survival of a plant after exposure to drought conditions; h) an increase in the extent of survival of a cell after exposure to drought conditions; i) an increase in the extent of survival of a plant after exposure to high salt conditions; and j) an increase in the extent of survival of a cell after exposure to high salt conditions.
- 59. The method of claim 58, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof.
- 60. The method of claim 58, wherein said CBF is selected from the group consisting SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, and fragments thereof.
- 61. The method of claim 58, wherein said nucleotide sequence specifically hybridizes under highly stringent conditions to SEQ ID NO: 1 or its complement under the conditions of 6×SSC and 65° C.
- 62. The method of claim 58, wherein said cell protectant is selected from the group consisting of proline and sucrose.
- 63. The method of claim 58, wherein said CBF comprises a sequence selected from the group consisting of:
(a) an AP2 domain comprising consecutive amino acid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102, and 104-106 of SEQ ID NO:2 are conserved in the AP2 domain of the CBF.
- 64. The method of claim 58, wherein said CBF is CBF3.
- 65. The method of claim 58, wherein said recombinant polynucleotide further comprises a regulatory region operably linked to the sequence encoding the CBF.
- 66. The method of claim 65, wherein said regulatory region is a constitutive promoter, an inducible promoter, a tissue specific promoter or a developmental stage specific promoter.
- 67. The method of claim 58, further comprising cold-acclimating said transformed cell.
- 68. A method of screening for a CBF, said method comprising:
(a) constructing yeast reporter strains by transforming yeast cells with a vector comprising C-repeat/DRE sequences as upstream activator sequence elements fused upstream of a reporter gene; (b) transforming said yeast reporter strains with an expression library containing random cDNA inserts each of which are fused to an activation domain; (c) growing said yeast reporter strains on differential media; and (c) screening for yeast colonies that comprise cDNA inserts encoding C-repeat/DRE binding domains fused to said activation domains.
- 69. The method of claim 68, wherein said reporter gene is a lacZ reporter gene, said reporter gene is operably linked to a GAL1 promoter, and said activation domain is a GAL4 activation domain;
wherein clones that contain a cDNA insert encoding a C-repeat/DRE binding domain fused to GAL4-activation domains bind upstream of the lacZ reporter gene carrying the wild type C-repeat/DRE sequence, activating transcription of the lacZ reporter gene. and wherein said differential media comprises X-gal treated filters, and said yeast colonies on said X-gal-treated filters are screened by their blue color.
- 70. The method of clain 68, wherein said CBF binds to a member of a class of DNA regulatory sequences that includes a subsequence selected from the group consisting of CCGAA, CCGAT, CCGAC, CCGAG, CCGTA, CCGTT, CCGTC, CCGTG, CCGCA, CCGCT, CCGCG, CCGCC, CCGGA, CCGGT, CCGGC, CCGGG, AACCG, ATCCG, ACCCG, AGCCG, TACCG, TTCCG, TCCCG, TGCCG, CACCG, CTCCG, CGCCG, CCCCG, GACCG, GTCCG, GCCCG, GGCCG, ACCGA, ACCGT, ACCGC, ACCGG, TCCGA, TCCGT, TCCGC, TCCGG, CCCGA, CCCGT, CCCGC, CCCGG, GCCGA, GCCGT, GCCGC, and GCCGG.
- 71. A method for identifying a CBF, said method comprising:
(a) identifying sequences by BLAST analysis in which ESTs in a database are queried using SEQ ID NO: 327, 328, 329, 330, or 331 as the query sequence (b) selecting positive EST sequences with an HSP BLAST score of at least 100 bits over at least 95% of the query sequence; and (c) organizing said positive EST sequences into contigs; thereby identifying a CBF sequence.
- 72. The method of claim 71, further comprising:
identifying complete predicted coding regions with a start and a stop codon; and cloning said CBF sequence directly from cDNA or genomic DNA by PCR using primers in the 5′ and 3′ flanking regions.
- 73. The method of claim 71, further comprising cloning said CBF sequence into a transformation vector.
- 74. The method of claim 73, wherein said transformation vector is pMEN65 or a transformation vector modified from pMEN65.
- 75. A method for enhancing the cold tolerance of a plant, said method comprising:
(a) transforming a plant with a recombinant polynucleotide comprising a nucleotide sequence encoding a CBF; (b) expressing the recombinant polynucleotide and (c) cold acclimating said transformed plant; wherein said cold acclimated transformed plant has an improved cold tolerance compared with a non-transformed plant that has been cold-acclimated; and wherein the enhanced cold tolerance is selected from the group consisting of: a) a decrease in the extent of a plant's injury; b) a decrease in the extent of a cell's injury; c) a decrease in the extent of a plant's growth inhibition; d) a decrease in the extent of a cell's growth inhibition; e) an increase in survival rate after exposure to cold temperatures; and f) an increase in survival rate after exposure to freezing temperatures.
- 76. The method of claim 75, wherein said plant is a rapeseed plant.
- 77. The method of claim 75, wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof.
- 78. The method of claim 75, wherein said CBF is selected from the group consisting SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, and fragments thereof.
- 79. The method of claim 75, wherein said nucleotide sequence specifically hybridizes under highly stringent conditions to SEQ ID NO: 1 or its complement under the conditions of 6×SSC and 65° C.
- 80. The method of claim 75, wherein said CBF comprises a sequence selected from the group consisting of:
(a) an AP2 domain comprising consecutive amino acid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102, and 104-106 of SEQ ID NO:2 are conserved in the AP2 domain of the CBF.
- 81. The method of claim 75, wherein the cold tolerance is enhanced by at least 2° C. compared with the cold tolerance of the non-transformed plant that has been cold-acclimated.
- 82. The method of claim 75, wherein the cold tolerance is enhanced by at least 5° C. compared with the cold tolerance of the non-transformed plant that has been cold-acclimated.
- 83. The method of claim 75, wherein the cold tolerance is enhanced by at least 8° C. compared with the cold tolerance of the non-transformed plant that has been cold-acclimated.
- 84. The method of claim 75, wherein said recombinant polynucleotide further comprises a regulatory region operably linked to the sequence encoding the CBF.
- 85. The method of claim 84, wherein said regulatory region is a constitutive promoter, an inducible promoter, a tissue specific promoter, or a developmental stage specific promoter.
- 86. The method of claim 85, wherein said inducible promoter is a cold-inducible promoter comprising a sequence selected from the group consisting of a sequence comprising nucleotides 1470-1520 of SEQ ID NO: 129 and a sequence comprising nucleotides 460-520 of SEQ ID NO: 130.
- 87. A method for altering the biomass of a plant, said method comprising:
(a) transforming said plant with a recombinant polynucleotide comprising a nucleotide sequence encoding a CBF; and (b) expressing said CBF in said transformed plant; wherein said transformed plant has a altered biomass compared with a plant that has not been transformed with said recombinant polynucleotide.
- 88. The method of claim 87, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof.
- 89. The method of claim 87, wherein said CBF is selected from the group consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, and fragments thereof.
- 90. The method of claim 87, wherein said nucleotide sequence specifically hybridizes under highly stringent conditions to SEQ ID NO: 1 or its complement under the conditions of 6×SSC and 65° C.
- 91. The method of claim 87, wherein said altered biomass is an increase in the leaf number, leaf size, or root mass of a plant.
- 92. The method of claim 87, wherein said CBF comprises a sequence selected from the group consisting of:
(a) an AP2 domain comprising consecutive amino acid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and 104-106 of SEQ ID NO:2 are conserved in the AP2 domain of the CBF.
- 93. The method of 87, wherein said CBF binds to a cold or dehydration transcription-regulating region comprising the sequence CCG.
- 94. The method of claim 87, wherein said CBF binds to a member of a class of DNA regulatory sequences which includes a subsequence selected from the group consisting of CCGAA, CCGAT, CCGAC, CCGAG, CCGTA, CCGTT, CCGTC, CCGTG, CCGCA, CCGCT, CCGCG, CCGCC, CCGGA, CCGGT, CCGGC, CCGGG, AACCG, ATCCG, ACCCG, AGCCG, TACCG, TTCCG, TCCCG, TGCCG, CACCG, CTCCG, CGCCG, CCCCG, GACCG, GTCCG, GCCCG, GGCCG, ACCGA, ACCGT, ACCGC, ACCGG, TCCGA, TCCGT, TCCGC, TCCGG, CCCGA, CCCGT, CCCGC, CCCGG, GCCGA, GCCGT, GCCGC, and GCCGG.
- 95. The method of claim 87, wherein said recombinant polynucleotide further comprises a regulatory region operably linked to the sequence encoding the CBF.
- 96. The method of claim 95, wherein said regulatory region is a constitutive promoter, an inducible promoter, a tissue specific promoter, or a developmental stage specific promoter.
- 97. The method of claim 87, wherein said plant is exposed to adverse environmental conditions, and said biomass is increased relative to an untransformed plant exposed to the same conditions.
- 98. The method of claim 87, wherein said plant cell is derived from a crop plant selected from the group consisting of Brassica juncea, Brassica napus, Brassica oleracea, Brassica rapa, Brassica rapa L, Brassica napus L, Glycine max, Raphanus sativus, Zea mays, Triticum, Oryza sativa, Secale cereale, Sorghum bicolor, Sorghum vulgare, and Hordeum vulgare.
- 99. A method for altering the biomass of a plant, said method comprising:
(a) transforming said plant with a recombinant polynucleotide comprising a nucleotide sequence encoding a CBF; wherein said CBF is sufficiently homologous to a consensus sequence shown in depicted in FIG. 19A, 19B, 19C, 19D, or 19E that the CBF is capable of binding to a CCG regulatory sequence and that the nucleotide sequence that encodes the CBF can hybridize to SEQ ID NO: 1 under the conditions of 6×SSC and 65° C.; and (b) expressing said CBF in said transformed plant; wherein said transformed plant has a altered biomass compared with a plant that has not been transformed with said recombinant polynucleotide.
- 100. A method for altering the biomass of a plant, said method comprising:
(a) altering the levels of a polynucleotide comprising a nucleotide sequence encoding a CBF in a plant; and (b) identifying a plant so altered; wherein said altered levels of said polynucleotide modifies the biomass of the plant compared to a plant that does not comprise said altered polynucleotide levels.
- 101. The method of claim 100, wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, 12, 18, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 70, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 115, 117, 119, 121, 123, 125, 127, orthologs, paralogs, and variants thereof.
- 102. The method of claim 100, wherein said CBF is selected from the group consisting of SEQ ID NO: 2, 10, 13, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 116, 118, 120, 122, 124, 126, 128, orthologs, paralogs, variants, and fragments thereof.
- 103. The method of claim 100, wherein said nucleotide sequence specifically hybridizes under highly stringent conditions to SEQ ID NO: 1 or its complement under the conditions of 6×SSC and 65° C.
- 104. The method of claim 100, wherein said altered biomass is an increase in leaf number, leaf size, or root mass.
- 105. The method of claim 100, wherein said CBF comprises a sequence selected from the group consisting of:
(a) an AP2 domain comprising consecutive amino acid residues 45-106 of SEQ ID NO: 2; (b) SEQ ID NO: 326; (c) SEQ ID NO: 325; or (d) SEQ ID NO: 321; wherein amino acid residues 45, 46, 48, 50-52, 54, 59, 60, 62, 64, 65, 67, 68, 71-73, 75-77, 79, 81, 83-91, 93-96, 99, 101, 102 and 104-106 of SEQ ID NO:2 are conserved in the AP2 domain of the CBF.
- 106. The method of claim 100, wherein said CBF binds to a cold or dehydration transcription-regulating region comprising the sequence CCG.
- 107. The method of claim 100, wherein said CBF binds to a member of a class of DNA regulatory sequences that includes a subsequence selected from the group consisting of CCGAA, CCGAT, CCGAC, CCGAG, CCGTA, CCGTT, CCGTC, CCGTG, CCGCA, CCGCT, CCGCG, CCGCC, CCGGA, CCGGT, CCGGC, CCGGG, AACCG, ATCCG, ACCCG, AGCCG, TACCG, TTCCG, TCCCG, TGCCG, CACCG, CTCCG, CGCCG, CCCCG, GACCG, GTCCG, GCCCG, GGCCG, ACCGA, ACCGT, ACCGC, ACCGG, TCCGA, TCCGT, TCCGC, TCCGG, CCCGA, CCCGT, CCCGC, CCCGG, GCCGA, GCCGT, GCCGC, and GCCGG.
- 108. The method of claim 100, wherein said recombinant polynucleotide further comprises a regulatory region operably linked to the sequence encoding the CBF.
- 109. The method of claim 108, wherein said regulatory region is a constitutive promoter, an inducible promoter, a tissue specific promoter, or a developmental stage specific promoter.
- 110. The method of claim 100, wherein said plant is exposed to adverse environmental conditions, and said biomass is increased relative to an untransformed plant exposed to the same conditions.
- 111. The method of claim 100, wherein said plant cell is derived from a crop plant selected from the group consisting of Brassica juncea, Brassica napus, Brassica oleracea, Brassica rapa, Brassica rapa L, Brassica napus L, Glycine max, Raphanus sativus, Zea mays, Triticum, Oryza sativa, Secale cereale, Sorghum bicolor, Sorghum vulgare, and Hordeum vulgare.
RELATED APPLICATION INFORMATION
[0001] This application is a continuation-in-part of the following U.S. applications: U.S. application Ser. No. 09/773,990, filed: Feb. 1, 2001, which in turn claimed priority from U.S. application Ser. No. 09/580,377, filed: May 26, 2000, which in turn claimed priority from U.S. provisional Application Serial No. 60/165,860, filed: Nov. 16, 1999, now abandoned; U.S. application Ser. No. 09/996,140, filed Nov. 26, 2001; U.S. application Ser. No. 09/601,802, filed: Sep. 15, 2000, which in turn claimed priority from PCT application No.: PCT/US99/01895, filed: Jan. 28, 1999, now abandoned, which in turn claimed priority from U.S. application Ser. No. 09/198,119, filed: Nov. 23, 1998, which issued as U.S. Pat. No. 6,417,428, which, in turn, claimed priority in part to U.S. application Ser. No. 09/018,233, filed: Feb. 3, 1998, now abandoned, U.S. application Ser. No. 09/017,816, filed: Feb. 3, 1998, now abandoned, U.S. application Ser. No. 09/018,235, filed: Feb. 3, 1998, now abandoned, U.S. application Ser. No. 09/017,575, filed: Feb. 3, 1998, now abandoned, U.S. application Ser. No. 09/018,227, filed: Feb. 3, 1998, now abandoned, and U.S. application Ser. No. 09/018,234, filed: Feb. 3, 1998, now abandoned, all six of which claimed priority in part to U.S. application Ser. No. 08/706,270, filed: Sep. 4, 1996, which issued as U.S. Pat. No. 5,892,009; U.S. application Ser. No. 09/627,348, filed Jul. 28, 2000, which in turn claimed priority from No. 60/148,200 filed Aug. 10, 1999, now abandoned, and U.S. provisional Application Serial No. 60/165,860 filed Nov. 16, 1999, now abandoned; U.S. application Ser. No. 09/713,994, filed: Nov. 16, 2000, which in turn claimed priority from No. 60/197,899 filed Apr. 17, 2000, now abandoned, U.S. provisional Application Serial No. 60/227,439 filed Aug. 22, 2000, now abandoned, and from U.S. provisional Application Serial No. 60/166,228 filed Nov. 17, 1999, now abandoned; each of which are incorporated herein by reference in their entirety.
Government Interests
[0002] This invention was supported by a subcontract under a USDA/CSREES Cooperative Agreement and an NSF/SBIR grant. The US Government has certain rights in this invention.
Provisional Applications (3)
|
Number |
Date |
Country |
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60197899 |
Apr 2000 |
US |
|
60227439 |
Aug 2000 |
US |
|
60166228 |
Nov 1999 |
US |
Continuation in Parts (14)
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Number |
Date |
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Parent |
09580377 |
May 2000 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
09773990 |
Feb 2001 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
09996140 |
Nov 2001 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
09601802 |
Sep 2000 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
PCT/US99/01895 |
Jan 1999 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
09198119 |
Nov 1998 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
09018233 |
Feb 1998 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
09017816 |
Feb 1998 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
09018235 |
Feb 1998 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
09017575 |
Feb 1998 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
09018227 |
Feb 1998 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
09018234 |
Feb 1998 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
08706270 |
Sep 1996 |
US |
Child |
10421138 |
Apr 2003 |
US |
Parent |
09713994 |
Nov 2000 |
US |
Child |
10421138 |
Apr 2003 |
US |