Production of gamma linolenic acid by a Δ 6-desaturase
Linoleic acid is converted into γ-linolenic acid by the enzyme Δ6-desaturase. The present invention is directed to isolated nucleic acids comprising the Δ6-desaturase gene. More particularly, the isolated nucleic acid comprises the promoter, coding region and termination regions of the Δ6-desaturase gene. The present invention provides recombinant constructions comprising the Δ6-desaturase coding region in functional combination with heterologous regulatory sequences. The nucleic acids and recombinant constructions of the instant invention are useful in the production of GLA in transgenic organisms.
1. A method of inducing production of octadecatetraeonic acid in a plant, which comprises transforming said plant with a nucleotide sequence encoding an evening primrose Δ6-desaturase.
2. The method of claim 1 , wherein said nucleotide sequence comprises at least one of the nucleotide sequence of SEQ ID NO: 26 or nucleotides 49 to 1401 of SEQ ID NO: 26.
3. The method of claim 1 , wherein said nucleotide sequence encodes SEQ ID NO: 27.
4. A method of inducing production of octadecatetraeonic acid in a plant, which comprises transforming said plant with an expression vector, wherein said vector comprises a nucleotide sequence encoding an evening primrose Δ6-desaturase.
5. The method of claim 4 , wherein said nucleotide sequence comprises at least one of the nucleotide sequence of SEQ ID NO: 26 or nucleotides 49 to 1401 of SEQ ID NO: 26.
6. The method of claim 4 , wherein said nucleotide sequence encodes SEQ ID NO: 27.
7. A method of inducing production of octadecatetraeonic acid in a plant, which comprises transforming said plant with an expression vector, wherein said vector comprises a promoter and a nucleotide sequence encoding an evening primrose Δ6-desaturase, and wherein said promoter is operably linked to said nucleotide sequence to effect expression of said evening primrose Δ6-desaturase.
8. The method of claim 7 , wherein said nucleotide sequence comprises at least one of the nucleotide sequence of SEQ ID NO: 26 or nucleotides 49 to 1401 of SEQ ID NO: 26.
9. The method of claim 7 , wherein said nucleotide sequence encodes SEQ ID NO: 27.
10. A method of inducing production of octadecatetraeonic acid in a plant, which comprises transforming said plant with an expression vector, wherein said vector comprises a promoter, a termination signal and a nucleotide sequence encoding an evening primrose Δ6-desaturase, and wherein said promoter and said termination signal are operably linked to said nucleotide sequence to effect expression of said evening primrose Δ6-desaturase.
11. The method of claim 10 , wherein said nucleotide sequence comprises at least one of the nucleotide sequence of SEQ ID NO: 26 or nucleotides 49 to 1401 of SEQ ID NO: 26.
12. The method of claim 10 , wherein said nucleotide sequence encodes SEQ ID NO: 27.
13. The method of any one of claims 7 - 12 , wherein said promoter is selected from a Δ6-desaturase promoter, an Anabaena carboxylase promoter, a helianthinin promoter, a glycinin in promoter, a napin promoter, the 35S promoter from CaMV, a helianthinin tissue-specific promoter, an oleosin seed-specific promoter, or an albumin seed-specific promoter.
14. The method of any one of claims 1 - 12 , wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant.
15. The method of claim 13 , wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant.
16. A method of inducing production of octadecatetraeonic acid in a plant, which comprises transforming said plant with a nucleotide sequence, wherein said nucleotide sequence encodes a plant Δ6-desaturase and hybridizes under stringency conditions to the complement of a polynucleotide sequence coding for an evening primrose Δ6-desaturase, wherein said polynucleotide sequence comprises SEQ ID NO: 26 or nucleotides 49 to 1401 of SEQ ID NO: 26, and wherein said stringency conditions comprise hybridization to filter-bound DNA in 6×SSC, 1× Denharts solution, 0.05% sodium pyrophosphate, 100 μg/ml denaturated salmon sperm DNA at 60° C., and washing in 4×, 2×, and 1×SET at 60° C.
17. A method of inducing production of octadecatetraeonic acid in a plant, which comprises transforming said plant with a nucleotide sequence, wherein said nucleotide sequence encodes a plant Δ6-desaturase and hybridizes under stringency conditions to the complement of a polynucleotide sequence molecule coding for a plant delta-6 desaturase which comprises
SEQ ID NO: 6,
SEQ ID NO: 12, and
SEQ ID NO: 20,
and wherein said stringency conditions comprise hybridization to filter-bound DNA in 6×SSC, 1× Denharts solution, 0.05% sodium pyrophosphate, 100 g/ml denaturated salmon sperm DNA at 60° C., and washing in 4×, 2×, and 1×SET at 60° C.
18. A method of inducing production of octadecatetraeonic acid in a plant, which comprises transforming said plant with a nucleotide sequence, wherein said nucleotide sequence encodes a plant Δ6-desaturase and hybridizes under stringency conditions to the complement of a polynucleotide sequence molecule coding for a plant delta-6 desaturase which comprises amino acids 159-164, 197-201 and 373-378 of SEQ ID NO: 27, and wherein said stringency conditions comprise hybridization to filter-bound DNA in 6×SSC, 1× Denharts solution, 0.05% sodium pyrophosphate, 100 μg/ml denaturated salmon sperm DNA at 60° C., and washing in 4×, 2×, and 1×SET at 60° C.
19. The method according to any one of claims 16 - 18 , wherein said nucleotide sequence that encodes said plant Δ6-desaturase is placed in an expression vector which comprises a promoter, and wherein said promoter is operably linked to said nucleotide sequence to effect expression of said plant Δ6-desaturase.
20. The method of claim 19 , wherein said promoter is selected from a Δ6-desaturase promoter, an Anabaena carboxylase promoter, a helianthinin promoter, a glycinin in promoter, a napin promoter, the 35S promoter from CaMV, a helianthinin tissue-specific promoter, an oleosm seed-specific promoter, or an albumin seed-specific promoter.
21. The method of any one of claims 16 - 18 , wherein said plant is a sunflower, soybean, maize, tobacco, peanut, carrot or oil seed rape plant.