Nucleic acids encoding anthelmintic agents and plants made therefrom
The present invention provides DNA constructs, transgenic plants containing such constructs, and methods of making the plants. The DNA constructs encode a polypeptide that when expressed results in the production of fatty acid compounds having anthelmintic activity. Transgenic plants expressing such a polypeptide can exhibit enhanced resistance to plant parasitic nematodes, particularly when expressed in vegetative tissues.
1. A transgenic plant containing at least one DNA construct, said construct comprising:
a) a nucleic acid encoding a polypeptide effective for catalysing the conversion of a substrate to a C16, C18, or C20 monounsaturated fatty acid products wherein said polypeptide has at least 95% sequence identity to the amino acid sequence shown in SEQ ID NO:41, wherein said fatty acid product has the following structure:
wherein X is hydrogen, CoA, glycerol, a monoglyceride, a diglyceride, ACP, methyl, Na+, phosphatidyicholine, or phosphatidylethanolamine, and wherein R 3 is C2, C4, or C6 alkyl; and
(b) a regulatory element operably linked to said nucleic acid encoding said polypeptide, wherein said regulatory element confers expression in a vegetative tissue of said plant; wherein the transgenic plant has anthelmintic activity.
2. The plant according to claim 1 , wherein the double bond between the 9 th and 10 th carbons is cis.
3. The plant according to claim 1 , wherein the double bond between the 9 th and 10 th carbons is trans.
4. The plant according to claim 1 , wherein said regulatory element is a 5′-regulatory element.
5. The plant of claim 4 , wherein said 5′-regulatory element confers expression in root tissue.
6. The plant of claim 5 , said plant having a significantly increased amount of a epoxy-fatty acid in roots of said plant relative to a corresponding plant that lacks said DNA construct.
7. The plant of claim 6 , wherein said epoxy-fatty acid is vernolic acid.
8. The plant of claim 7 , wherein said vernolic acid constitutes from about 0.1% to about 35% of the total fatty acid content of said roots.
9. The plant of claim 4 , wherein said 5′-regulatory element is selected from the group consisting of a potato ribosomal protein S27a Ubi3 promoter, a RB7 promoter, an alfalfa histone H3.2 promoter, an IRT2 promoter, an Arabidopsis FAD2 5′-UTR, an Arabidopsis FAD3 5′-UTR, a Ubi3 5′-UTR, an alfalfa histone H3.2 5′-UTR, and a CaMV35S 5′-UTR.
10. The plant of claim 1 , wherein said regulatory element comprises a first 5′-regulatory element operably linked to a second 5′-regulatory element, wherein said first 5′-regulatory element is an Ubi3 promoter and said second 5′-regulatory element is selected from the group consisting of an Arabidopsis FAD2 5′-UTR, an Arabidopsis FAD3 5′-UTR, a potato ribosomal protein S27a 5′-UTR, a Ubi3 5′-UTR, and a CaMV35S 5′-UTR.
11. The plant of claim 4 , wherein said DNA construct further comprises a 3′-regulatory element.
12. The plant of claim 11 , wherein said 3′-regulatory element comprises a Ubi3 terminator or an E9 pea terminator.
13. The plant of claim 11 , wherein said 5′-regulatory element is selected from the group consisting of an Arabidopsis FAD2 5′-UTR and an Arabidopsis FAD3 5′-UTR and said 3′-regulatory element is selected from the group consisting of an Arabidopsis FAD2 3′-UTR and an Arabidopsis FAD3 3′-UTR.
14. The plant of claim 13 , wherein said 5′-regulatory element comprises SEQ ID NOS: 43 or 44 and said 3′-regulatory element comprises SEQ ID NO: 45.
15. The plant of claim 1 , wherein said at least one DNA construct further comprises at least one regulatory element that confers expression in vegetative tissues of a plant operably linked to a nucleic acid that encodes a PDAT or DAGAT polypeptide.
16. The plant of claim 1 , said plant further comprising a second DNA construct, said second DNA construct comprising at least one regulatory element that confers expression in vegetative tissues of a plant operably linked to a nucleic acid that encodes a PDAT or DAGAT polypeptide.
17. The plant of claim 1 , wherein R 3 is C2 alkyl or C4 alkyl.
18. The plant of claim 1 , wherein said nucleic acid encoding said polypeptide has the nucleotide sequence shown in SEQ ID NO: 32.
19. The plant of claim 1 , where said plant is selected from the group consisting of tobacco, tomato, soybean, corn, cotton, rice, wheat, banana, carrot, potato, strawberry and turf grass.
20. A method of making a transgenic plant having anthelmintic activity, said method comprising introducing a construct into a plant, wherein said construct comprises:
a) a nucleic acid encoding a polypeptide effective for catalysing the conversion of a substrate to a C16, C18, or C20 monounsaturated fatty acid product, wherein said polypeptide has the amino acid sequence shown in SEQ ID NO:41, wherein said fatty acid product has the following structure:
wherein X is hydrogen, CoA, glycerol, a monoglyceride, a diglyceride, ACP, methyl, Na+, phosphatidyicholine, or phosphatidylethanolamine, and wherein R 3 is C2, C4, or C6 alkyl; and
(b) a regulatory element operably linked to said nucleic acid encoding said polypeptide, wherein said regulatory element confers expression in a vegetative tissue of said plant.
21. The method of claim 20 , wherein said regulatory element of said construct is a 5′-regulatory element.
22. The method of claim 21 , wherein said 5′-regulatory element comprises a potato ribosomal protein S27a Ubi3 promoter, a RB7 promoter, an alfalfa histone H3.2 promoter, an IRT2 promoter, an Arabidopsis FAD2 5′-UTR, an Arabidopsis FAD3 5′-UTR, a Ubi3 5′-UTR, an alfalfa histone H3.2 5′-UTR, and a CaMV35S 5′-UTR.
23. The method of claim 21 , wherein said regulatory element comprises a first 5′-regulatory element operably linked to a second 5′-regulatory element, wherein said first 5′-regulatory element is an Ubi3 promoter and said second 5′-regulatory element is selected from the group consisting of an Arabidopsis FAD2 5′-UTR, an Arabidopsis FAD3 5′-UTR, a potato ribosomal protein S27a 5′-UTR, a Ubi3 5′-UTR, and a CaMV35S 5′-UTR.
24. The method of claim 21 , wherein said DNA construct further comprises a 3′-regulatory element.
25. The method of claim 24 , wherein said 5′-regulatory element comprises SEQ ID NO: 43 or SEQ ID NO: 44 and said 3′-UTR comprises SEQ ID NO: 45.
26. An isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:32.
27. A recombinant nucleic acid construct comprising at least one regulatory element that confers expression in a vegetative tissue of a plant, said regulatory element operably linked to a nucleic acid having the nucleotide sequence shown in SEQ ID NO:32.
28. The nucleic acid construct of claim 27 , wherein said at least one regulatory element comprises a 5′-regulatory element having the nucleotide sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44.
29. The nucleic acid construct of claim 28 , wherein said construct further comprises a 3′-regulatory element having the nucleotide sequence set forth in SEQ ID NO: 45.
30. A transgenic plant harboring a DNA construct comprising a nucleic acid encoding a fatty acid epoxygenase polypeptide operably linked to a regulatory element conferring expression of said polypeptide in a vegetative tissue of said plant, wherein said polypeptide has the amino acid sequence shown in SEQ ID NO:41.
31. The plant of claim 30 , said plant having a significantly increased amount of a epoxy-fatty acid in roots of said plant relative to a corresponding plant that lacks said DNA construct.
32. The plant of claim 31 , wherein said epoxy-fatty acid is vemolic acid.
33. The plant of claim 32 , wherein said vernolic acid constitutes from about 0.1% to about 35% of the total fatty acid content of said roots.