Brassica or helianthus plants having mutant delta-12 or delta-15 sequences

• US 7,135,614 B1
• Filed: 12/14/1995
• Issued: 11/14/2006
• Est. Priority Date: 08/30/1990
• Status: Expired due to Fees

• Current Assignee: Cargill, Inc. (Agricultural Services Division)
• Original Assignee: Cargill, Inc. (Agricultural Services Division)
• Inventors: Miao, Guo-Hua, DeBonte, Lorin R., Fan, Zhegong
• Primary Examiner(s): McElwain, Elizabeth F.
• Application Number:
  US08/572,027
• Time in Patent Office: 3,988 Days
• Field of Search: 800/205, 800/264, 800/270, 800/306, 800/298, 800/320.1, 800/261, 800/281, 536/27.1, 536/236, 435/22, 435/23, 435/65, 435/412, 435/424, 435/430
• US Class Current: 800/264
• 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)

Abstract

Plants are disclosed that contain a mutation in a delta-12 or delta-15 fatty acid desaturase gene. Preferred plants are rapeseed and sunflower plants. Plants carrying such mutant genes have altered fatty acid composition in seeds. In one embodiment, a plant contains a mutation in a region having the conserved motif His-Xaa-Xaa-His, found in delta-12 and delta-15 fatty acid desaturases. A preferred motif has the sequence His-Glu-Cys-Gly-His. A preferred mutation in this motif has the amino acid sequence His-Lys-Cys-Gly-His. Nucleic acid fragments are disclosed that comprise a sequence of at least 20 nucleotides from a mutant delta-12 or delta-15 fatty acid desaturase gene.

Claims

1. An isolated nucleic acid comprising a full-length Brassicaceae delta-15 fatty acid desaturase coding sequence having at least one mutation in a region of said desaturuse coding sequence encoding a His-Xaa-Xaa-Xaa-His amino acid motif, wherein said at least one mutation renders the product of said desaturase coding sequence non-functional and wherein said sequence includes said at least one mutation.

2. The nucleic acid fragment of claim 1, wherein said sequence encodes a microsomal gene product.

3. The nucleic acid fragment of claim 1, wherein said at least one mutation introduces a non-conservative amino acid substitution in said region.

4. The nucleic acid fragment of claim 3, wherein the wild-type amino acid sequence of said motif comprises the sequence His-Asp-Cys-Gly-His (SEQ ID NO:

9).

5. The nucleic acid fragment of claim 4, wherein said at

6. The nucleic acid fragment of claim 1, wherein said mutant desaturase coding sequence is from a Brassica napus plant.

7. A Brassicacea plant containing a full-length coding sequence of a delta-15 fatty acid desaturase gene having at least one mutation, wherein said at least one mutation is in a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif and wherein said mutation renders the product of said desaturase gene non-functional.

8. The plant of claim 7, wherein said mutation confers a decreased level of α

-linolenic acid in seeds of said plant.

9. The plant of claim 7, wherein said mutant desaturase gene encodes a microsomal gene product.

10. The plant of claim 7, wherein said at least one mutation comprises a non-conservative amino acid substitution in said region.

11. The plant of claim 10, wherein the wild-type amino acid sequence of said motif comprises the sequence His-Asp-Cys-Gly-His (SEQ ID NQ:

9).

12. The plant of claim 11, wherein said at least one mutation comprises a codon encoding Lys in place of the codon encoding Asp.

13. The plant of claim 7, wherein said mutant desaturase gene is from a Brassica napus plant.

14. The plant of claim 7, wherein said plant is a Brassica napus plant.

15. A Brassicacea plant containing:

a) a full-length coding sequence from a delta-12 fatty acid desaturane gene having at least one mutation, said at least one delta-12 gene mutation in a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif; and

b) a full-length coding sequence from a delta-15 fatty acid desaturase gene having at least one mutation, said at least one delta-15 gene mutation in a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif,

wherein said delta-12 gene mutation and said delta-15 gene mutation render the products of said delta-12 desaturase gene and said delia-15 desaturase gene, respectively, non-functional.

16. The plant of claim 15, wherein said mutant genes confer a decreased level of α

-linolenic acid in seeds of said plant compared to α

-linolenic acid levels in corresponding seeds lacking said mutant genes.

17. A Brassicaceae or Helianthus plant containing a full-length coding sequence of a delta-12 fatty acid desaturase gene having at least one mutation, said at least one mutation in a region encoding a Tyr-Leu-Asn-Asn-Pro (SEQ ID NO:

50) amino acid motif and wherein said mutation renders the product of said desaturase gene non-functional.

18. A method for producing a Brossicaceat or Helionihus plant line, comprising the steps of:

a) inducing mutagenesis in cells of a starting variety of a Brassicaceae or Helianthus species;

b) obtaining one or more plants from said cells;

c) identifying at least one of said plants that contains a delta-12 fatty acid desaturase gene having at least one mutation, said at least one mutation in a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif, wherein said mutation in said delta-12 gene renders the product of said delta-12 desaturase gene non-functional; and

d) producing said Brassicaceae or Helianthus plant line from said at least one plant, said Brassicaceae or Helianthus plant line having said at least one mutation in said delta-12 gene.

19. The method of claim 18, wherein said plant line yields an oil having a stabilized linoleic acid content from about 2.0% to about 12.0%.

20. The method of claim 18, further comprising the steps of:

e) inducing mutagenesis in cells of said Brassicaceae or Helianthus plant line;

f) obtaining one or more plants from said cells of said Brassicaceae or Helianthus plant line;

g) identifying at least one of said plants from step f) that contains a delta-15 fatty acid desaturase gene having at least one mutation, wherein said at least one mutation in said delta-15 gene is in a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif, wherein said mutation renders the product of said delta-15 desaturase gene non-functional; and

h) producing a second Brassicaceae or Helianthus plant line from said at least one plant identified in step g), said second plant line having said at least one mutation in said delta-12 gene and said at least one mutation in said delta-15 gene.

21. The method of claim 18, wherein said starting variety is a Brassica napus variety.

22. The method of claim 21, wherein said mutation is in a first form of delta-12 fatty acid desaturase.

23. The method of claim 22, further comprising the step of crossing a plant of said plant line to a plant having a mutation in a second form of delta-12 fatty acid desaturase.

24. The method of claim 23, wherein said second mutation is in a region other than a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif.

25. The method of claim 22, further comprising the steps of:

e) inducing mutagenesis in cells of said Brassicaceae or Helianthus plant line;

f) obtaining one or more plants from said cells of said Brassicaceae or Helianthus plant line;

g) identifying at least one of said plants from step f) that contains a second delta-12 fatty acid desaturase gene having at least one mutation, said second gene mutation in a region other than a region encoding a His-Xaa-Xaa-Xaa-LJis amino acid motif; and

h) producing a second Brassicaceae or Helianthus plant line from said at least one plant identified in step g), said second Brassicaceae or HeIianthus plant line having said first and second delta-12 gene mutations.

26. The method of claim 18, wherein said identifying step comprises a technique selected from the group consisting of:

PCR, 3SR and direct polynucleotide sequencing.

27. A method for producing a Brassicaceae plant line, comprising the steps of:

a) inducing mutagenesis in cells of a starting variety of a Brassicaceac species;

b) obtaining one or more plants from said cells;

c) identifying at least one of said plants that contains a delta-15 fatty acid desaturase gene having at least one mutation, said at least one mutation in a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif, wherein said at least one mutation renders the product of said delta-15 desaturase gene non-functional; and

d) producing said Brassicaceae plant line from said at least one plant, said Brassicaceae plant line having said mutation in said delta-15 gene.

28. The method of claim 27, wherein said identifying step comprises a technique selected from the group consisting of:

PCR, 3SR and direct polynucleotide sequencing.

29. An isolated nucleic acid comprising a full length Brassicaceae or Helianthus delta-12 fatty acid desaturase coding sequence having at least one mutation in a region of said desaturase coding sequence encoding a Tyr-Leu-Asn-Asn-Pro (SEQ ID NO:

50) amino acid motif, wherein said at least one mutation renders the product of said desaturase coding sequence non-functional and wherein said sequence includes said at least one mutation.

34. The nucleic acid fragment of claim 29, wherein said at least one mutation comprises a codon encoding His in place of the codon encoding Leu.

30. A method for identifying a mutation in a Brassicaceae plant, comprising:

a) providing a Brassicaceae plant having a decreased α

-linolenic acid content as compared with a corresponding control Brassicaceae plant; and

b) identifying at least one mutation inn delta-15 fatty acid desaturase gene of said plant, said at least one mutation in a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif, wherein said mutation renders the product of said delta-15 fatty acid desaturase gene non-functional.

31. The method of claim 30, wherein said identifying step comprises a technique selected from the group consisting of:

PCR, 3SR and direct polynucleotide sequencing.

32. A method for identifying a mutation in a Brassicaceae or Helianthus plant, comprising:

a) providing a Brassicaceae or Helianthus plant having an increased oleic acid content as compared with a corresponding control Brassicaceae or Helianthus plant; and

b) identifying at least one mutation in a delta-12 fatty acid desaturase gene of said plant, said at least one mutation in a region encoding a His-Xaa-Xaa-Xaa-His amino acid motif, wherein said mutation renders the product of said delta-12 fatty acid desaturase gene non-functional.

33. The method of claim 32, wherein said identifying step comprises a technique selected from the group consisting of:

PCR., 3SR and direct polynucleotide sequencing.