Fatty acid desaturase genes from plants

• US 5,952,544 A
• Filed: 08/26/1994
• Issued: 09/14/1999
• Est. Priority Date: 12/04/1991
• 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: Wierzbicki, Anna M., Browse, John, Grau, Luis Perez, Pierce, John W. Jr., Yadav, Narendra S., Kinney, Anthony J.
• Primary Examiner(s): McElwain, Elizabeth F.
• Application Number:
  US08/244,205
• Time in Patent Office: 1,845 Days
• Field of Search: 536/23.6, 435/172.3, 435/69.1, 435/320.1, 435/240.4, 435/419, 800/205, 800/DIG. 69, 426/601
• US Class Current: 800/295
• 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 encoding a plant plastid or microsomal delta-15 fatty acid desaturase enzyme, which catalyzes a reaction at C15-C16 of a fatty acyl chain and further 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, 6X SSC, 5X Denhardt'"'"'s, 0.5% sodium dodecyl sulfate (SDS), 100 μ

g denatured calf thymus DNA at 50°

C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, repeat with 2X SSC, 0.5% SDS at 45°

C. for 30 min, then repeat twice with 0.2X SSC, 0.5% SDS at 50°

C. for 30 min each;

(b) hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt'"'"'s, 0.5% sodium dodecyl sulfate (SDS), 100 μ

g denatured calf thymus DNA at 50°

C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, repeat with 2X SSC, 0.5% SDS at 45°

C. for 30 min, then repeat twice with 0.2X SSC, 0.5% SDS at 60°

C. for 30 min each;

(c) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50°

C. for eighteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, then washing for 5 min at 50°

C. in 0.2X SSPE, 1% SDS;

(d) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50°

C. for sixteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, then wash with fresh solution for 10 min, then wash for 5 min at 50°

C. in 0.5X SSPE, 1% SDS;

(e) hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt'"'"'s, 0.5% sodium dodecyl sulfate (SDS), 100 μ

g denatured calf thymus DNA at 50°

C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, then wash twice with 2X SSC, 0.5% SDS at 45°

C. for 30 min each and then wash twice with 0.2X SSC, 0.5% SDS at 60°

C. for 30 min each;

or

(f) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50°

C. for eighteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, followed by washing for 5 min at 50°

C. in 0.2X SSPE, 1% SDS.

5. An isolated nucleic acid fragment of claim 1, claim 2, claim claim 3 or claim 4 wherein said fragment is isolated from a plant selected from the group consisting of soybean, oilseed Brassica species, Arabidopsis thaliana and corn.

6. A chimeric gene capable of causing altered levels of linolenic acid in a transformed plant cell, the gene comprising a nucleic acid fragment of any of claims 1, claim 2, claim 3 or claim 4 the fragment operably linked to suitable regulatory sequences.

7. Plants containing the chimeric gene of claim 6.

8. A method of producing seed oil containing altered levels of linolenic (18:

3) acid comprising;

(a) transforming a plant cell of an oil-producing species with a chimeric gene of claim 5;

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

(c) screening progeny seeds from the fertile plants of step (b) for the desired levels of linolenic (18;

3) acid; and

(d) processing the progeny seed of step (c) to obtain seed oil containing altered levels of linolenic (18;

3) acid.

9. A method of claim 8 wherein said plant cell of an oil-producing species is selected from the group consisting of Arabidopsis thaliana, soybean, oilseed Brassica napus, sunflower, cotton, peanut, and corn.

14. The method of claim 8 wherein said plant cell of an oil-producing species is selected from the group consisting of Arabidopsis thaliana, soybean, oilseed Brassica napus, sunflower, cotton and corn.

2. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a plant plastid or microsomal enzyme which catalyzes the formation of a double bond between carbon positions 3 and 4 numbered from the methyl end of a fatty acyl chain, and further wherein the amino acid sequence comprising said enzyme contains at least one of the following amino acid sequences selected from the group consisting of FVLGHDCGHGSF, GHDCGH, HDIGTHVIHHLFP, HDIGTH, or HVIHHL.

3. An isolated nucleic acid fragment encoding a plant plastid or microsomal enzyme which catalyzes the formation of a double bond between carbon positions 3 and 4 numbered from the methyl end of a fatty acyl chain, wherein said isolated nucleic acid fragment encodes a protein comprising any one of the amino acid sequences set forth in SEQ ID NOS:

2, 5, 7, 9, 11, 12, 15 or 17.

4. An isolated nucleic acid fragment encoding an enzyme which catalyzes a reaction at C15-C16 of a fatty acyl chain wherein said isolated nucleic acid fragment hybridizes to the isolated nucleic acid fragment of claim 3 under one of the following sets of conditions:

(a) hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt'"'"'s, 0.5% sodium dodecyl sulfate (SDS), 100 μ

g denatured calf thymus DNA at 50°

C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, repeat with 2X SSC, 0.5% SDS at 45°

C. for 30 min, then repeat twice with 0.2X SSC, 0.5% SDS at 50°

C. for 30 min each;

(b) hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt'"'"'s, 0.5% sodium dodecyl sulfate (SDS), 100 μ

g denatured calf thymus DNA at 50°

C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, repeat with 2X SSC, 0.5% SDS at 45°

C. for 30 min, then repeat twice with 0.2X SSC, 0.5% SDS at 60°

C. for 30 min each;

(c) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50°

C. for eighteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, then washing for 5 min at 50°

C. in 0.2X SSPE, 1% SDS;

(d) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50°

C. for sixteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, then wash with fresh solution for 10 min, then wash for 5 min at 50°

C. in 0.5X SSPE, 1% SDS;

(e) hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt'"'"'s, 0.5% sodium dodecyl sulfate (SDS), 100 μ

g denatured calf thymus DNA at 50°

C. overnight and wash with 6X SSC, 0.5% SDS at room temperature for 15 min, then wash twice with 2X SSC, 0.5% SDS at 45°

C. for 30 min each and then wash twice with 0.2X SSC, 0.5% SDS at 60°

C. for 30 min each;

or

(f) hybridization in 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 1% sodium dodecyl sulfate (SDS), 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA at 50°

C. for eighteen hours and wash twice at room temperature with 2X SSPE, 1% SDS for 5 min, followed by washing for 5 min at 50°

C. in 0.2X SSPE, 1% SDS.

10. The isolated genomic DNA of Arabidopsis thaliana comprising the microsomal delta-15 desaturase identified by accession number ATCC 75167.

11. A plasmid designated pXF1 and bearing accession number ATCC 68874 comprising the DNA sequence of SEQ ID NO:

10 which encodes a soybean delta-15 desaturase enzyme.

12. A plasmid designated pBNSF3 and bearing accession number ATCC 68854 comprising the DNA sequence of SEQ ID NO:

6 which encodes an oilseed Brassica species delta-15 desaturase enzyme.

13. An isolated Polymerase Chain Reaction Product designated PCR20 comprising the DNA sequence of SEQ ID NO:

14 which encodes a portion of the Zea mays delta-15 desaturase enzyme.