Claims
- 1. An isolated nucleic acid sequence encoding a divinyl ether synthase.
- 2. A nucleic acid sequence encoding a tomato divinyl ether synthase.
- 3. A nucleic acid sequence according to claim 2, wherein the tomato is Lycopersicon esculentum.
- 4. A nucleic acid sequence according to claim 1, wherein the sequence encoding a divinyl ether synthase comprises SEQ ID NO: 1.
- 5. An isolated nucleic acid sequence that hybridizes under conditions of low stringency to a nucleic sequence comprising SEQ ID NO: 1.
- 6. An isolated nucleic acid sequence that hybridizes under conditions of high stringency to a nucleic sequence comprising SEQ ID NO: 1.
- 7. A nucleic acid sequence according to claim 5, wherein the sequence encodes a polypeptide that catalyzes the conversion of hydroperoxide fatty acids to divinyl ether fatty acid that contain an oxygen within the hydrocarbon chain.
- 8. An isolated nucleic acid sequence encoding a divinyl ether synthase, wherein said synthase competes for binding to a dihydroperoxide fatty acid substrate with a protein encoded by a nucleic acid sequence comprising SEQ ID NO: 1.
- 9. An isolated antisense sequence corresponding to a nucleic acid sequence encoding a divinyl ether synthase.
- 10. A nucleic acid sequence according to claim 1 operably linked to a heterologous promoter.
- 11. A vector comprising a nucleic acid sequence according to claim 1.
- 12. A vector comprising a nucleic acid sequence according to claim 10.
- 13. A purified polypeptide encoded by the nucleic acid sequence of claim 1.
- 14. A purified divinyl ether synthase.
- 15. A purified tomato divinyl ether synthase.
- 16. A purified divinyl synthase according to claim 15, wherein the tomato is Lycopersicon esculentum.
- 17. A purified polypeptide comprising amino acid SEQ ID NO:2.
- 18. A composition, comprising:
a) at least one purified divinyl ether fatty acid; and b) a carrier suitable for applying the fatty acid to a plant, a plant part, or a plant seed.
- 19. A composition according to claim 18, wherein the divinyl ether fatty acid is a product of divinyl ether synthase when incubated with hydroperoxide fatty acid substrates selected from the group consisting of mono-, di-, and tri-hydroxy fatty acids fatty acids with variable chain length and from none to one or more additional functional group selected from the group consisting of acetylenic bonds, conjugated acetylenic and ethylenic bonds, allenic groups, cyclopropane, cyclopropene, cyclopentene and furan rings, epoxy-, hydroxy- and keto-groups and double bonds of both the cis and trans configuration and separated by more than one methylene group, wherein the substrates are either free fatty acids, or fatty acids incorporated into a larger molecule.
- 20. A composition according to claim 19, wherein the divinyl ether fatty acids are selected from the group consisting of colneleic acid and colnelenic acid.
- 21. A composition according to claim 18, wherein the composition further comprises at least one component which aids plant growth and protection.
- 22. A composition according to claim 21, wherein the component is selected from the group consisting of fertilizer, insecticide, fungicide, nematocide, and herbicide.
- 23. A composition according to claim 18, wherein the composition further comprises at least one component which facilitates application of the composition to the plant, the plant part or the plant seed.
- 24. A composition according to claim 23, wherein the component is selected from the group consisting of buffering agents, wetting agents, coating agents, and abrading agents.
- 25. A composition according to claim 18, wherein the composition further comprises at least one component which aids seed storage and germination.
- 26. A composition according to claim 25, wherein the component is selected from the group consisting of clays and polysaccharides.
- 27. An organism transformed with a heterologous gene encoding a divinyl ether synthase.
- 28. An organism according to claim 27, wherein the gene is derived from tomato.
- 29. An organism according to claim 28, wherein the tomato is Lycopersicon esculentum.
- 30. An organism according to claim 29, wherein the gene comprises SEQ ID NO: 1.
- 31. An organism co-transformed with a first heterologous gene encoding a divinyl ether synthase and with a second heterologous gene encoding a lipoxygenase.
- 32. An organism co-transformed with a heterologous gene encoding a fusion polypeptide comprising a divinyl ether synthase and a lipoxygenase.
- 33. An organism according to claim 27, where the organism is selected from the group consisting of plants and microorganisms.
- 34. A plant transformed with a heterologous gene encoding a divinyl ether synthase.
- 35. A plant cell transformed with a heterologous gene encoding a divinyl ether synthase.
- 36. A plant seed transformed with a heterologous gene encoding a divinyl ether synthase.
- 37. Oil from the transgenic plant of claim 34.
- 38. A method for expressing a synthase in a plant, comprising:
a) providing:
i) plant tissue, and ii) a vector comprising a nucleic acid sequence encoding a divinyl ether synthase; and b) transfecting the plant tissue with the vector under conditions such that the synthase is expressed.
- 39. A method for expressing an antisense sequence in a plant, comprising:
a) providing:
i) plant tissue; and ii) a vector comprising an antisense sequence corresponding to a nucleic acid sequence encoding a divinyl ether synthase; and b) transfecting the plant tissue with the vector under conditions such that the antisense sequence is expressed.
- 40. A method for producing a variant of divinyl ether synthase comprising:
a) providing a nucleic acid sequence encoding a divinyl ether synthase; and b) mutagenizing the nucleic acid sequence so as to produce a variant.
- 41. A method according to claim 40, further comprising:
c) screening the variant for activity.
- 42. A method of producing divinyl ether fatty acids in vitro, comprising:
a) providing a purified divinyl ether synthase and at least one fatty acid substrate of the enzyme; and b) incubating the synthase with the substrate under conditions such that divinyl ether fatty acid is produced.
- 43. A method according to claim 42, wherein the substrate selected from the group consisting of mono-, di-, and tri-hydroperoxide fatty acids with variable chain length and from none to one or more additional functional group selected from the group consisting of acetylenic bonds, conjugated acetylenic and ethylenic bonds, allenic groups, cyclopropane, cyclopropene, cyclopentene and furan rings, epoxy-, hydroxy- and keto-groups and double bonds of both the cis and trans configuration and separated by more than one methylene group, wherein the substrates are either free fatty acids, or fatty acids incorporated into a larger molecule.
- 44. A method according to claim 43, wherein the fatty acid substrates are selected from the group consisting of 9-hydroperoxystearidonic acid, 13-hydroperoxystearidonic acid, 9-hydroperoxylinoleic acid, 9-hydroperoxylinolenic acid, 13-hydroperoxylinoleic acid, 13 hydroperoxylinolenic acid, 9-hydroperoxyarachidonic acid, and 13-hydroperoxyarachidonic acid.
- 45. A method of producing divinyl ether fatty acids in vitro, comprising:
a) providing an isolated nucleic sequence encoding a divinyl ether synthase and at least on fatty acid substrate of the enzyme; and b) incubating the sequence with the substrate in a inscription/translation system under conditions such that the sequence is expressed and divinyl ether fatty acids are produced.
- 46. A method according to claim 45, wherein the fatty acid substrate is selected from the group consisting of mono-, di-, and tri-hydroperoxy fatty acids with variable chain length and from none to one or more additional functional group selected from the group consisting of acetylenic bonds, conjugated acetylenic and ethylenic bonds, allenic groups, cyclopropane, cyclopropene, cyclopentene and furan rings, epoxy-, hydroxy- and keto-groups and double bonds of both the cis and trans configuration and separated by more than one methylene group, wherein the substrates are either free fatty acids, or fatty acids incorporated into a larger molecule.
- 47. A method according to claim 46, wherein the substrate is selected from the group consisting of 9-hydroperoxystearidonic acid, 13-hydroperoxystearidonic acid, 9-hydroperoxylinoleic acid, 9-hydroperoxylinolenic acid, 13-hydroperoxylinoleic acid, 13 hydroperoxylinolenic acid, 9-hydroperoxyarachidonic acid, and 13-hydroperoxyarachidonic acid.
- 48. A method of producing divinyl ether fatty acids in vitro, comprising:
a) providing a purified divinyl ether synthase, a purified lipoxygenase and at least one fatty acid substrate of the lipoxygenase; and b) incubating the synthase and the lipoxygenase with the substrate under conditions such that divinyl ether fatty acid is produced.
- 49. A method according to claim 48, wherein the fatty acid substrate is selected from the group consisting of polyunsaturated fatty acids with at least two double bonds in a 1Z, 4Z-pentadiene structure and with variable chain length and from none to more than one additional functional group selected from the group consisting of acetylenic bonds, conjugated acetylenic and ethylenic bonds, allenic groups, cyclopropane, cyclopropene, cyclopentene and furan rings, epoxy-, hydroxy- and keto-groups and double bonds of both the cis and trans configuration and separated by more than one methylene group, wherein the substrates are either free fatty acids, or fatty acids incorporated into a larger molecule.
- 50. A method according to claim 49, wherein the substrate is selected from the group consisting of stearidonic acid, linoleic acid, linolenic acid and arachidonic acid.
- 51. A method of producing divinyl ether fatty acids by fermentation, comprising:
a) providing a microorganism transformed with a heterologous gene encoding a divinyl ether synthase and at least one substrate of the synthase; and b) incubating the microorganism with the substrate under conditions such that divinyl ether fatty acids are produced.
- 52. A method according to claim 51, wherein the fatty acid substrate is selected from the group consisting of mono-, di-, and tri-hydroperoxide fatty acids with variable chain length and from none to one or more additional functional group selected from the group consisting of acetylenic bonds, conjugated acetylenic and ethylenic bonds, allenic groups, cyclopropane, cyclopropene, cyclopentene and furan rings, epoxy-, hydroxy- and keto-groups and double bonds of both the cis and trans configuration and separated by more than one methylene group, wherein the substrates are either free fatty acids, or fatty acids incorporated into a larger molecule, such as a glycerolipid.
- 53. A method of producing divinyl ether fatty acids by fermentation, comprising:
a) providing a microorganism co-transformed with a first heterologous gene encoding a divinyl ether synthase and with a second heterologous gene encoding a lipoxygenase and at least one substrate of the lipoxygenase; and b) incubating the microorganism with the substrate under conditions such that divinyl ether fatty acid is produced.
- 54. A method according to claim 53, wherein the fatty acid substrate is selected from the group consisting of polyunsaturated fatty acids with at least two double bonds in a 1Z, 4Z-pentadiene structure and with variable chain length and from none to more than one additional functional group selected from the group consisting of acetylenic bonds, conjugated acetylenic and ethylenic bonds, allenic groups, cyclopropane, cyclopropene, cyclopentene and furan rings, epoxy-, hydroxy- and keto-groups and double bonds of both the cis and trans configuration and separated by more than one methylene group, wherein the substrates are either free fatty acids, or fatty acids incorporated into a larger molecule.
- 55. A method of producing divinyl ether fatty acids by fermentation, comprising:
a) providing a microorganism transformed with a heterologous gene encoding a fission polypeptide comprising a divinyl ether synthase and a lipoxygenase and at least one substrate of the lipoxygenase; and b) incubating the microorganism with the substrate under conditions such that divinyl ether fatty acid is produced.
- 56. A method according to claim 55, wherein the fatty acid substrate is selected from the group consisting of polyunsaturated fatty acids with at least two double bonds in a 1Z, 4Z-pentadiene structure and with variable chain length and from none to one or more additional functional group selected from the group consisting of acetylenic bonds, conjugated acetylenic and ethylenic bonds, allenic groups, cyclopropane, cyclopropene, cyclopentene and furan rings, epoxy-, hydroxy- and keto-groups and double bonds of both the cis and trans configuration and separated by more than one methylene group, wherein the substrates are either free fatty acids, or fatty acids incorporated into a larger molecule.
- 57. A method of producing divinyl ether fatty acids in a plant comprising:
a) providing a plant and a heterologous gene encoding a divinyl ether synthase; and b) transforming the plant with the heterologous gene such that divinyl ether fatty acid is produced.
- 58. A method of producing divinyl ether fatty acids in a plant comprising: growing a plant transformed with a heterologous gene encoding a divinyl ether synthase under conditions such that divinyl ether fatty acid is produced.
- 59. A method of producing divinyl ether fatty acids in a plant comprising:
a) providing a plant, a first heterologous gene encoding a divinyl ether synthase, and a second heterologous gene encoding a lipoxygenase; and b) co-transforming the plant with the first heterologous gene and with the second heterologous gene such that divinyl ether fatty acid is produced.
- 60. A method of producing divinyl ether fatty acids in a plant comprising growing a plant transformed with a first heterologous gene encoding a divinyl ether synthase, and with a second heterologous gene encoding a lipoxygenase under conditions such that divinyl ether fatty acid is produced.
- 61. A method of producing divinyl ether fatty acids in a plant comprising:
a) providing a plant and a heterologous gene encoding a fusion polypeptide comprising a divinyl ether synthase and a lipoxygenase; and b) transforming a plant with the heterologous gene, such that divinyl ether fatty acids are produced.
- 62. A method of producing divinyl ether fatty acids in a plant comprising growing a plant with a heterologous gene encoding a fusion polypeptide comprising a divinyl ether synthase and a lipoxygenase under conditions such that divinyl ether fatty acids are produced.
- 63. A method of protecting a plant from a pathogen comprising growing a plant transformed with a heterologous gene encoding a divinyl ether synthase under conditions such that the plant is protected from the pathogen.
- 64. A method of protecting a plant from a pathogen comprising growing a plant transformed with a first heterologous gene encoding a divinyl ether synthase and with a second heterologous gene encoding a lipoxygenase under conditions such that the plant is protected from the pathogen.
- 65. A method of protecting a plant from a pathogen comprising growing a plant transformed with a heterologous gene encoding a fusion polypeptide comprising a divinyl ether synthase and a lipoxygenase under conditions such that the plant is protected from the pathogen.
- 66. A method according to claim 63, wherein the heterologous gene encoding a divinyl ether synthase is under control of a promoter that controls expression of divinyl ether fatty acids in a specific tissue of the plant.
- 67. A method of claim 63, wherein the heterologous gene encoding a divinyl ether synthase is under control of a promoter that controls expression of divinyl ether fatty acids in a specific developmental phase of the plant.
- 68. A method of claim 63, wherein the heterologous gene encoding a divinyl ether synthase is under control of a promoter that is inducible.
- 69. A method of claim 63, where the plant is a potato plant, and the pathogen is Phyotphora infestans.
- 70. A method of protecting a plant from a pathogen comprising:
a) providing a transgenic plant transformed with a heterologous gene encoding a divinyl ether synthase; and b) growing the transgenic plant under conditions effective to protect the plant from the pathogen.
- 71. A method of protecting a plant from a pathogen comprising:
a) providing a transgenic seed transformed with a heterologous gene encoding a divinyl ether synthase; and b) growing a transgenic plant from the transgenic seed under conditions effective to protect the plant from the pathogens.
- 72. A method of protecting a plant from a pathogen comprising:
1. providing a transgenic plant material transformed with a heterologous gene encoding a divinyl ether synthase; and 2. growing a transgenic plant from the transgenic plant material under conditions effective to protect the plant from the pathogen.
- 73. A method of protecting a plant from a pathogen comprising applying a composition comprising at least one purified divinyl ether fatty acid to a plant, a plant part, or a plant seed, such that the plant, the plant part, or the plant seed is protected from the pathogen.
- 74. A method according to claim 73, wherein the divinyl ether fatty acid is a product of divinyl ether synthase when incubated with hydroperoxide fatty acid substrates selected from the group consisting of mono-, di-, and tri-hydroxy fatty acids fatty acids with variable chain length and from none to one or more additional functional group selected from the group consisting of acetylenic bonds, conjugated acetylenic and ethylenic bonds, allenic groups, cyclopropane, cyclopropene, cyclopentene and furan rings, epoxy-, hydroxy- and keto-groups and double bonds of both the cis and trans configuration and separated by more than one methylene group, wherein the substrates are either free fatty acids, or fatty acids incorporated into a larger molecule.
- 75. A method according to claim 64, wherein the divinyl ether fatty acids are selected from the group consisting of colneleic acid and colnelenic acid.
- 76. A method according to claim 73, wherein the composition further comprises at least one additional component, wherein the component is selected from the group consisting of a carrier suitable for applying the fatty acid to a plant, a plant part, or a plant seed, a component which aids plant growth and protection, and a component which facilitates application of the composition to the plant, the plant part, or the plant seed.
- 77. A method for screening for divinyl ether synthases comprising:
a) providing a candidate divinyl ether synthase; and b) analyzing the amino acid sequence of said candidate divinyl ether synthase for the presence of the motif AGxxAF in the I-helix consensus sequence.
- 78. A computer readable medium encoding a representation of the nucleic acid sequence of any of claims 1-8.
- 79. A computer readable medium encoding a representation of the polypeptide of any of claims 13-17.
- 80. A method of catalyzing a reaction, comprising combining a purified divinyl ether synthase and a hydroperoxy substrate under conditions effective to convert the hydroperoxy substrate to a divinyl ether product.
- 81. A method of catalyzing a reaction, comprising combining a transgenic organism and a hydroperoxy substrate under conditions effective to convert the hydroperoxy substrate to a divinyl ether product, wherein the transgenic organism comprises a heterologous gene encoding a divinyl ether synthase.
Parent Case Info
[0001] This application claims priority to U.S. Provisional Application Serial No. 60/238,415, filed on Oct. 6, 2000.
Government Interests
[0002] This invention was made in part during work partially supported by the United States Department of Energy grant no. 61-4224 and United States Department of Agriculture grant no. 61-3200-010. The United States government may have certain rights in the invention.
PCT Information
Filing Document |
Filing Date |
Country |
Kind |
PCT/US01/31296 |
10/5/2001 |
WO |
|