Specific for palmitoyl, stearoyl and oleoyl-alp thioesters nucleic acid fragments encoding acyl-acp thiosesterase enzymes and the use of these fragments in altering plant oil composition

• US 5,945,585 A
• Filed: 10/09/1997
• Issued: 08/31/1999
• Est. Priority Date: 12/20/1990
• Status: Expired due to Fees

• 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: Yadav, Narendra S., Hitz, William Dean
• Primary Examiner(s): Smith, Lynette F.
• Assistant Examiner(s): Nelson, Amy J.
• Application Number:
  US08/948,176
• Time in Patent Office: 691 Days
• Field of Search: 435/172.3, 435/370.1, 435/419, 435/468, 536/23.2, 536/23.6, 536/24.3, 536/24.5, 800/205, 800/250, 800/DIG. 26, 800/281, 800/286, 800/298, 800/312
• US Class Current: 800/312
• CPC Class Codes: C11B 1/00 Production of fats or fatty oils from raw materials
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12P 7/6463 obtained from glyceride producing microorganisms, e.g. single cell oil

Abstract

Isolated nucleic acid fragments encoding an acyl-ACP thioesterase enzyme which catalyzes the hydrolysis of palmitoyl, stearoyl and oleoyl-ACP thioesters are described. Use of such fragments in altering plant oil composition is also described.

Claims

1. An isolated nucleic acid fragment encoding an acyl-ACP thioesterase which catalyzes the hydrolysis of palmitoyl, stearoyl and oleoyl-ACP thioesters, wherein said isolated nucleic acid fragment hybridizes to nucleotides 271 to 1206 of SEQ ID NO:

1 or nucleotides 282 to 1217 of SEQ ID NO;

3 or nucleotides 210 to 1121 of SEQ ID NO;

23 under one of the following sets of conditions;

(a) hybridization in 6×

SSC, 1 mM EDTA, 1% SDS, and 100 μ

g/mL denatured salmon sperm DNA at 45°

C. for 10 h and wash 3 times at room temperature with 0.6×

SSC containing 0.1% SDS, then once at 50°

C. for 5 min in the same solution and two additional washes for 5 min each at 50°

C. in 0.2×

SSC, 0.1% SDS followed by a 1 min rinse under the same conditions;

or

(b) hybridization in 6×

SSC, 1 mM EDTA, 1% SDS, and 100 μ

g/mL denatured salmon sperm DNA at 45°

C. for 10 h and wash twice at room temperature with 0.6×

SSC containing 0.1% SDS followed by two 5 minute washes and one 1 minute wash in the same solution and all at 52°

C.

3. The isolated nucleic acid fragment of claims 1 or 2 wherein said fragment is isolated from the group consisting of soybean and Brassica napus.

7. A chimeric gene causing altered levels of acyl-ACP thioesterase activity in a transformed plant cell, the gene comprising the isolated nucleic acid fragment of claim 1 operably linked either in the sense or the antisense orientation to regulatory sequences.

8. A chimeric gene causing reduced levels of mature seed acyl-ACP thioesterase activity in a transformed soybean plant the gene comprising the nucleic acid fragment of claim 1 operably linked in the sense orientation to regulatory sequences wherein expression of the nucleic acid fragment results in co-suppression of endogenous thioesterase activity.

9. A chimeric gene causing increased levels of mature seed acyl-ACP thioesterase activity in a transformed plant, the gene comprising the isolated nucleic acid fragment of claims 1 or 2 operably linked to regulatory sequences.

10. A chimeric gene causing reduced levels of mature seed acyl-ACP thioesterase activity in a transformed soybean plant, the gene comprising the nucleic acid fragment of claim 1 operably linked in the antisense orientation to regulatory sequences wherein expression of the nucleic acid fragment results in antisense inhibition of endogenous thioesterase activity.

11. A chimeric gene causing increased levels of mature seed acyl-ACP thioesterase activity in a transformed plant, the gene comprising the nucleic acid fragment of claim 1 operably linked in the sense orientation to regulatory sequences.

12. A plant cell transformed with the chimeric gene of claim 7.

13. A transformed soybean plant having lowered levels of acyl-ACP thioesterase activity comprising an endogenous acyl-ACP thioesterase gene and the chimeric gene of claim 8.

14. A method of producing seed oil comprising altered levels of palmitic and stearic acids comprising:

(a) transforming plant cells of an oil-producing species with the chimeric gene of claim 7 to produce transformed plant cells;

(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 palmitic and stearic acids; and

(d) processing the progeny seeds of step (c) to obtain oil comprising altered levels of palmitic and stearic acids as compared to seeds of untransformed plants.

15. The method of claim 14 wherein the oil-producing species is selected from the group consisting of soybean, oil seed Brassica species, sunflower, cotton, cocoa, peanut, safflower, and corn.

16. A method of producing soybean seed oil comprising reduced levels of palmitic and stearic acids comprising:

(a) transforming soybean cells with the chimeric gene of claim 8 to produce transforming soybean cells;

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

(c) screening progeny seeds from the fertile soybean plants of step (b) for the desired reduced levels of palmitic and stearic acids; and

(d) processing the progeny seeds of step (c) to obtain oil comprising reduced levels of palmitic and stearic acids as compared to seeds of untransformed plants.

18. A method of producing plant oil comprising increased levels of palmitic and stearic acids comprising:

(a) transforming plant cells of an oil-producing species with the chimeric gene of claim 11 to produce transformed plant cells;

(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 increased levels of palmitic and stearic acids; and

(d) processing the progeny seeds of step (c) to obtain oil comprising increased levels of palmitic and stearic acids as compared to seeds of untransformed plants.

2. An isolated nucleic acid fragment of at least 41 base pairs, which is useful in antisense inhibition or cosuppression of endogenous thioesterase activity in a transformed plant, wherein said isolated nucleic acid fragment hybridizes to nucleotides 271 to 1206 of SEQ ID NO:

1 or nucleotides 282 to 1217 of SEQ ID NO;

3 under the following set of conditions;

hybridization in 6×

SSC, 1 mM EDTA, 1% SDS, and 100 μ

g/mL denatured salmon sperm DNA at 45°

C. for 10 h and wash 3 times at room temperature with 0.6×

SSC containing 0.1% SDS, then once at 50°

C. for 5 min in the same solution and two additional washes for 5 min each at 50°

C. in 0.2×

SSC, 0.1% SDS followed by a 1 min rinse under the same conditions.

4. An isolated nucleic acid fragment encoding the Brassica species acyl-ACP thioesterase encoded by nucleotides 1 to 1412 of SEQ ID NO:

23.

5. An isolated nucleic acid fragment encoding the Brassica species acyl-ACP thioesterase encoded by nucleotides 36 to 1121 of SEQ ID NO:

23.

6. An isolated nucleic acid fragment encoding the Brassica species acyl-ACP thioesterase encoded by nucleotides 210 to 1121 of SEQ ID NO:

23.

17. A method of producing plant seed oil comprising reduced levels of palmitic and stearic acids comprising:

(a) transforming plant cells of an oil-producing species with a nucleotide sequence selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

3, and SEQ ID NO;

23, operably linked in the antisense orientation to transcription regulatory sequences, to produce transformed plant cells;

(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 reduced levels of palmitic and stearic acids; and

(d) processing the progeny seeds of step (c) to obtain oil comprising reduced levels of palmitic and stearic acids as compared to seeds of untransformed plants.