Limnanthes oil genes

• US 20040224413A1
• Filed: 05/27/2004
• Published: 11/11/2004
• Est. Priority Date: 03/20/1998
• Status: Active Grant

• Current Assignee: Anthony J. Kinney, Edgar B. Cahoon, Steven J. Vollmer, William D. Hitz
• Original Assignee: Anthony J. Kinney, Edgar B. Cahoon, Steven J. Vollmer, William D. Hitz
• Inventors: Hitz, William D., Kinney, Anthony J., Cahoon, Edgar B., Vollmer, Steven J.
• Granted Patent: US 7,495,149 B2
• Time in Patent Office: Days
• US Class Current: 435/468
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/0083 Miscellaneous (1.14.99)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)

Abstract

This invention relates to an isolated nucleic acid fragment encoding an enzyme involved in lipid biosynthesis. The invention also relates to the construction of a chimeric gene encoding all or a portion of the enzyme involved in lipid biosynthesis, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the enzyme involved in lipid biosynthesis in a transformed host cell.

Claims

1. An isolated nucleic acid fragment encoding all or a substantial portion of a delta-5 acyl-CoA desaturase comprising a member selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO;

2;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO;

2; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

2. The isolated nucleic acid fragment of claim 1 wherein the nucleotide sequence of the fragment comprises all or a portion of the sequence set forth in SEQ ID NO:

1.

3. A chimeric gene comprising the nucleic acid fragment of claim 1 operably linked to suitable regulatory sequences.

4. A transformed host cell comprising the chimeric gene of claim 3.

11. A method of altering the level of expression of an enzyme involved in lipid biosynthesis in a host cell comprising:

(a) transforming a host cell with the chimeric gene of any of claims 3 and 8; and

(b) growing the transformed host cell produced in step (a) under conditions that are suitable for expression of the chimeric gene

wherein expression of the chimeric gene results in production of altered levels of an enzyme involved in lipid biosynthesis in the transformed host cell.

12. A method of producing a desaturated fatty acid comprising a double bond in the delta-5 position in a host cell, the method comprising:

(a) transforming a host cell with the chimeric gene of claim 3;

and

(b) growing the transformed host cell produced in step (a) under conditions that are suitable for expression of the chimeric gene

wherein expression of the chimeric gene results in production of a desaturated fatty acid comprising a double bond in the delta-5 position.

17. A method of producing seed oil comprising a desaturated fatty acid wherein the fatty acid comprises a double bond in the delta-5 position, the method comprising:

(a) transforming a plant cell with the chimeric gene of claim 3;

(b) growing a fertile plant from the transformed plant cell of step (a);

(c) obtaining a seed from the plant of step (b); and

(d) processing the seed of step (c) to obtain oil

wherein the oil comprises a desaturated fatty acid wherein the fatty acid comprises a double bond in the delta-5 position.

18. The method of claim 17 wherein the plant cell is derived from an oilseed crop.

19. The method of claim 18 wherein the oilseed crop is soybean.

20. The seed oil produced by the method of claim 17.

31. A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding an enzyme involved in lipid biosynthesis comprising:

(a) probing a cDNA or genomic library with the nucleic acid fragment of any of claims 1 and 6;

(b) identifying a DNA clone that hybridizes with the nucleic acid fragment of any of claims 1, and 6;

(c) isolating the DNA clone identified in step (b); and

(d) sequencing the cDNA or genomic fragment that comprises the clone isolated in step (c)

wherein the sequenced nucleic acid fragment encodes all or a substantial portion of the amino acid sequence encoding an enzyme involved in lipid biosynthesis.

33. The product of the method of claim 31.

5. A delta-5 acyl-CoA desaturase polypeptide comprising all or a substantial portion of the amino acid sequence set forth in SEQ ID NO:

2.

6. An isolated nucleic acid fragment encoding all or a substantial portion of a fatty acyl-CoA elongase comprising a member selected from the group consisting of:

(a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO;

5 and SEQ ID NO;

7;

(b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO;

5 and SEQ ID NO;

7; and

(c) an isolated nucleic acid fragment that is complementary to (a) or (b).

7. The isolated nucleic acid fragment of claim 6 wherein the nucleotide sequence of the fragment comprises all or a portion of the sequence set forth in a member selected from the group consisting of SEQ ID NO:

4 and SEQ ID NO;

6.

8. A chimeric gene comprising the nucleic acid fragment of claim 6 operably linked to suitable regulatory sequences.

9. A transformed host cell comprising the chimeric gene of claim 8.

21. A method of reducing the level of 16 carbon fatty acids in a host cell comprising:

(a) transforming a host cell with the chimeric gene of claim 8;

and

(b) growing the transformed host cell produced in step (a) under conditions that are suitable for expression of the chimeric gene

wherein expression of the chimeric gene results in the reduction of the level of 16 carbon fatty acids in the transformed host cell when compared to the level of 16 carbon fatty acids in a host cell that is not transformed with the chimeric gene of claim 8.

22. A method of producing seed oil with reduced levels of 16 carbon fatty acids comprising:

(a) transforming a plant cell with the chimeric gene of claim 8;

(b) growing a fertile plant from the transformed plant cell of step (a);

(c) obtaining a seed from the plant of step (b); and

(d) processing the seed of step (c) to obtain oil

wherein the oil comprises a lower level of 16 carbon fatty acids than oil obtained from a seed obtained from a plant that was grown from a plant cell that was not transformed with the chimeric gene of claim 8.

23. The method of claim 22 wherein the plant is an oilseed crop.

24. The method of claim 23 wherein the oilseed crop is soybean.

25. The seed oil produced by the method of claim 22.

26. A method of increasing the level of 20 carbon fatty acids in a host cell comprising:

(a) transforming a host cell with the chimeric gene of claim 8;

and

(b) growing the transformed host cell produced in step (a) under conditions that are suitable for expression of the chimeric gene

wherein expression of the chimeric gene results in an increase in the level of 20 carbon fatty acids in the transformed host cell when compared with the level of 20 carbon fatty acids in host cells that are not transformed with the chimeric gene of claim 8.

27. A method of producing seed oil with increased levels of 20 carbon fatty acids comprising:

(e) transforming a plant cell with the chimeric gene of claim 8;

(f) growing a fertile plant from the transformed plant cell of step (a);

(g) obtaining a seed from the plant of step (b); and

(h) processing the seed of step (c) to obtain oil

wherein the oil comprises a higher level of 20 carbon fatty acids than oil obtained from a seed obtained from a plant that was grown from a plant cell that was not transformed with the chimeric gene of claim 8.

28. The method of claim 27 wherein the plant is an oilseed crop.

29. The method of claim 28 wherein the oilseed crop is soybean.

30. A seed oil produced by the method of claim 27.

10. A fatty acyl-CoA elongase polypeptide comprising all or a substantial portion of the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NO:

5 and SEQ ID NO;

7.

13. A seed obtained from an oilseed crop wherein the seed comprises a desaturated fatty acid wherein the fatty acid comprises a double bond in the delta-5 position.

14. The seed of claim 13 wherein the oilseed crop is soybean.

15. An oil obtained from the seed of an oilseed crop wherein the oil comprises a desaturated fatty acid wherein the fatty acid comprises a double bond in the delta-5 position.

16. The oil of claim 15 wherein the oilseed crop is soybean.

32. A method of obtaining a nucleic acid fragment encoding a substantial portion of an amino acid sequence encoding an enzyme involved in lipid biosynthesis comprising:

(a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence set forth in any of SEQ ID NOs;

1, 4 and 6; and

(b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector

wherein the amplified nucleic acid fragment encodes a substantial portion of an amino acid sequence encoding an enzyme involved in lipid biosynthesis.

34. The product of the method of claim 32.