Genes for microsomal delta-12 fatty acid desaturases and hydroxylases from plants

• US 6,372,965 B1
• Filed: 08/14/1998
• Issued: 04/16/2002
• Est. Priority Date: 11/17/1992
• Status: Expired due to Term

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Perez-Grau, Luis, Hitz, William, Okuley, John Joseph, Lightner, Jonathan Edward, Yadav, Narendra S., Kinney, Anthony John
• Primary Examiner(s): McElwain, Elizabeth F.
• Application Number:
  US09/133,962
• Time in Patent Office: 1,341 Days
• Field of Search: 800/281, 800/298, 435/69.1, 435/468, 435/419, 435/320.1, 536/23.6
• US Class Current: 800/298
• 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: C12Y 114/19006 DELTA12-fatty-acid desaturase (1.14.19.6), i.e. oleoyl-CoA DELTA12 desaturase

Abstract

The preparation and use of nucleic acid fragments encoding fatty acid desaturase enzymes are described. The invention permits alteration of plant lipid composition. Chimeric genes incorporating such nucleic acid fragments with suitable regulatory sequences may be used to create transgenic plants with altered levels of unsaturated fatty acids.

Claims

1. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a plant enzyme selected from the group consisting of a delta-12 desaturase and a delta-12 hydroxylase wherein said isolated nucleic acid fragment hybridizes to one of the nucleotide sequences set forth in SEQ ID NOS:

1, 3, 5, 7, 9, 11, and 15 under one of the following sets of conditions;

(a) hybridization in 50 mM Tris, pH 7.6, 6×

SSC, 5×

Denhardt'"'"'s, 0.5% sodium dodecyl sulfate (SDS), 100 μ

g denatured calf thymus DNA at 50°

C. and wash twice with 2×

SSC, 0.5% SDS at room temperature for 15 min each, then wash twice with 0.2×

SSC, 0.5% SDS at room temperature for 15 min each and then wash twice with 0.2×

SSC, 0.5% SDS at 50°

C. for 15 min each;

(b) hybridization in 6×

SSPE, 5×

Denhardt'"'"'s solution, 0.5% sodium dodecyl sulfate (SDS), 5% dextran sulfate, 100 μ

g denatured calf thymus DNA at 50°

C. and wash twice with 2×

SSC, 0.5% SDS at room temperature for 15 min each, then wash twice with 0.2×

SSC, 0.5% SDS at room temperature for 15 min each and then wash twice with 0.2×

SSC, 0.5% SDS at 50°

C. for 15 min each;

or

(c) hybridization in 50% formamide, 5×

SSPE, 1% sodium dodecyl sulfate (SDS), 1% Denhardt'"'"'s Reagent, 100 μ

g denatured salmon sperm DNA at 42°

C. and wash twice with 2×

SSPE, 0.2% SDS at 42°

C. for 15 min each, then wash twice with 0.2×

SSPE, 0.2% SDS at 55°

C. for 30 min each.

2. The isolated nucleic acid fragment of claim 1 wherein said fragment is isolated from an oil-producing plant species.

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

3. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a delta-12 fatty acid hydroxylase.

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

6. Plants comprising in their genome the chimeric gene of claim 4 or claim 5.

10. Seeds of the plant of claim 6.

7. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding an enzyme selected from the group consisting of a delta-12 desaturase and a delta-12 hydroxylase wherein the amino acid sequence comprising said enzyme contains at least one of the following amino acid sequences selected from the group consisting of:

AIPPHCF, AWXXYW, HECGH, LLVPY, WKYSHR, and SHRRHH.

11. The isolated nucleic acid fragment of claim 7, 8 or 9 wherein said fragment is isolated from an oil-producing plant species.

12. The chimeric gene comprising the nucleic acid fragment of claim 7, 8 or 9 operably linked to suitable regulatory sequences.

13. The isolated nucleic acid fragment of claim 7, 8 or 9 comprising a nucleic acid sequence encoding a plant microsomal delta-12 fatty acid desaturase.

8. An isolated nucleic acid fragment encoding an enzyme selected from the group consisting of a delta-12 desaturase and a delta-12 hydroxylase wherein said isolated nucleic acid fragment encodes a protein comprising any one of the amino acid sequences set forth in SEQ ID NOS:

2, 4, 6, 8, 10 or 12.

9. An isolated nucleic acid fragment encoding an enzyme selected from the group consisting of a delta-12 desaturase and a delta-12 hydroxylase wherein said isolated nucleic acid fragment hybridizes to the isolated nucleic acid fragment of claim 8 under one of the following sets of conditions:

(a) hybridization in 50 mM Tris, pH 7.6, 6×

SSC, 5×

Denhardt'"'"'s, 0.5% sodium dodecyl sulfate (SDS), 100 μ

g denatured calf thymus DNA at 50°

C. and wash twice with 2×

SSC, 0.5% SDS at room temperature for 15 min each, then wash twice with 0.2×

SSC, 0.5% SDS at room temperature for 15 min each and then wash twice with 0.2×

SSC, 0.5% SDS at 50°

C. for 15 min each;

(b) hybridization in 6×

SSPE, 5×

Denhardt'"'"'s solution, 0.5% sodium dodecyl sulfate (SDS), 5% dextran sulfate, 100 μ

g denatured calf thymus DNA at 50°

C. and wash twice with 2×

SSC, 0.5% SDS at room temperature for 15 min each, then wash twice with 0.2×

SSC, 0.5% SDS at room temperature for 15 min each and then wash twice with 0.2×

SSC, 0.5% SDS at 50°

C. for 15 min each;

or

(c) hybridization in 50% formamide, 5×

SSPE, 1% sodium dodecyl sulfate (SDS), 1% Denhardt'"'"'s Reagent, 100 μ

g denatured salmon sperm DNA at 42°

C. and wash twice with 2×

SSPE, 0.2% SDS at 42°

C. for 15 min each, then wash twice with 0.2×

SSPE, 0.2% SDS at 55°

C. for 30 min each.