Cloning of △~6-Fatty Acid Desaturase Gene cDNA from Oenothera Biennis Using Technology of Race

Abstract: Polyunsaturated fatty acids (PUFAs) are valuable products because of their involvement in several aspects of human health care.At present,market demand for polyunsaturated fatty acids such asγ-Lionlenic acid(GLA) is growing continually and current sources are inadequate for satisfying this demand.Therefore seeking for alternative source are demanding.△~6-fatty acid desaturase is the rate-limiting enzyme for the biosynthesis of PUFAs.A 565bp eDNA fragment was amplified from developing seeds of wild plant Oenothera biennis whose seeds are rich in high activity GLA with degenerate oligonucleotide primers designed based on the sequences information for plants A6-fatty acid desaturase genes via RT-PCR and sequencesed.Gene specific primers derived from this partial sequence were used for the amplification of the 3′and 5′ends of the eDNA by RACE method,and this lead to a 753bp fragment of 3′and 313bp fragement of 5′ends.These three fragments were spliced to be a 1445bp fragment which contains complete 3′noncoding region.We blasted the amino acid sequence that the nucleotide sequence encodes in Genebank database.The result shows it is high identical with△~6-fatty acid desaturase gene inω-6 pathway in homology.Obtaining the 1445bp fragment successfully was prepared for the next step experiment,which is to clone full-length cDNA of Oenothera biennis△~6-fatty acid desaturase gene and express it specifically in seeds of transgenic oil crops,and regard transgenic oil crops as bioreactor of PUFAs…
Key words: Polyunsaturated fatty acids (PUFAs); cDNA ofΔ~6-fatty acid desaturase; Oenothera biennis ; 3’RACE; 5’RACE

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