Claims
- 1. A method of identifying a triterpene biosynthesis coding sequence comprising:
(a) obtaining a cell from a target legume species; (b) contacting said cell with methyl jasmonate; and (c) identifying a coding sequence which is specifically upregulated in the cell following the contacting with methyl jasmonate to identify a triterpene biosynthesis gene.
- 2. The method of claim 1, further comprising screening a polypeptide encoded by the coding sequence for the ability to catalyze a step in triterpene biosynthesis.
- 3. The method of claim 1, wherein the target legume is selected from the group consisting of soybean, alfalfa, Medicago truncatula, peanuts, beans, peas, lentils, Lotus japonicus, chickpea, cowpea, lupin, vetch, Sophora species, Acacia species, licorice and clover.
- 4. The method of claim 3, wherein the target legume is Medicago truncatula.
- 5. The method of claim 1, wherein the cell is grown in a tissue culture.
- 6. The method of claim 5, wherein the tissue culture is a cell suspension culture.
- 7. The method of claim 1, wherein the cell is obtained from a plant treated with said methyl jasmonate.
- 8. The method of claim 1, wherein the step of obtaining a cell is further defined as comprising obtaining a population of cells from the target legume.
- 9. The method of claim 8, comprising preparing a tissue culture from the cell.
- 10. The method of claim 1, wherein the step of identifying a coding sequence is further defined as comprising identifying a plurality of coding sequences specifically upregulated in said cell relative to the corresponding coding sequences in one or more other cells which have not been contacted with methyl jasmonate.
- 11. The method of claim 1, wherein the step of identifying a coding sequence comprises obtaining an RNA transcribed by the coding sequence and/or a cDNA derived therefrom.
- 12. The method of claim 11, further comprising the steps of:
(a) labeling said RNA and/or cDNA; and (b) hybridizing the labeled RNA or cDNA to an array comprising a plurality of coding sequences from the target legume.
- 13. The method of claim 10, further comprising preparing an array comprising the RNA transcripts or cDNAs thereof arranged on a support material.
- 14. The method of claim 1, wherein identifying a coding sequence further comprises selecting a coding sequence having homology to a cytochrome P450.
- 15. The method of claim 13, wherein identifying a coding sequence further comprises selecting a coding sequence having homology to glycosyltransferase.
- 16. The method of claim 1, wherein identifying a coding sequence further comprises selecting a coding sequence having homology to a squalene synthase.
- 17. The method of claim 1, wherein identifying a coding sequence further comprises selecting a coding sequence having homology to a squalene epoxidase.
- 18. The method of claim 1, wherein identifying a coding sequence further comprises selecting a coding sequence having homology to β-amyrin synthase.
- 19. The method of claim 1, wherein identifying a coding sequence comprises use of subtractive hybridization.
- 20. The method of claim 1, wherein identifying a coding sequence comprises use of nucleic acid sequencing.
- 21. The method of claim 1, wherein identifying a coding sequence comprises use of RT-PCR.
- 22. The method of claim 1, wherein identifying a coding sequence comprises use of differential display.
- 23. The method of claim 1, wherein identifying a coding sequence comprises use of an array.
- 24. The method of claim 1, wherein screening comprises transforming a host cell with the coding sequence and determining the ability of the host cell to catalyze a step in triterpene biosynthesis.
- 25. The method of claim 24, further comprising contacting the host cell with a substrate of said step in triterpene biosynthesis.
- 26. The method of claim 25, wherein the substrate is selected from the group consisting of farnesyl diphosphate, squalene, oxidosqualene and β-amyrin.
- 27. The method of claim 25, wherein the substrate is selected from the group consisting of bayogenin, hederagenin, medicagenic acid, soyasapogenol B and soyasapogenol E.
- 28. The method of claim 24, wherein the host cell is a yeast cell.
- 29. The method of claim 24, wherein the host cell is a plant cell.
- 30. The method of claim 29, further comprising regenerating a plant from the plant cell.
- 31. The method of claim 24, wherein the host cell is a bacterial cell.
BACKGROUND OF THE INVENTION
[0001] This application claims the priority of U.S. provisional patent application serial No. 60/380,159, filed May 4, 2002, the entire disclosure of which is specifically incorporated herein by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60380159 |
May 2002 |
US |