Cloning and Expression of the Δ~4 Fatty Acid Desaturase Gene from Thraustochytrium sp.FJN10

Abstract: Docosahexaenoic acid (DHA) is a valuable polyunsaturated fatty acids (PUFAs) due to its involvement in several aspects of human health care, and the research on DHA resource and biosynthesis has drawn much attention.In order to explain the mechanism of DHA biosynthesis in marine fungi and construct the strain or plant producing DHA highly, the fatty acid desaturase gene in Thraustochytrium sp. FJN10 was cloned and expressed. And the major research is as follow: (1) FJN-10 was isolated by using pine pollen as a bait, there were vegetative cell, sporangium and zoospores in its life cycle; It produces long chain polyunsaturated fatty acid highly, and there is about 43.2% DHA in its total fatty acids; Its 18S rRNA gene was isolated by PCR and sequenced (GenBank accession No. AY773276), and the phylogenetic tree based on 18S rRNA gene indicated that FJN-10 was the closest to Thraustochytrium sp MBIC11092. (2) The total RNA was isolated from FJN-10 cultured in the optional temperature and time; And the full-length cDNA of delta-4 fatty acid desaturase was obtained by RT-PCR and RACE ( rapid amplification of cDNA ends ), its whole ORF is 1560 bp encoding 519 amino acids; The analysis of genomic sequence showed that Thraustochytrium sp. FJN10 delta-4 fatty acid desaturase gene has no introns; (3) To elucidate the function of this novel putative desaturse, a recombinant plasmid pYFAD4 was constructed, and it was subsequently transformed into Saccaromyces cerevisiae strain INVScl for heterologous expression, as a result, a novel peak corresponding toω6 Docosapentaenoic acid (DPA, 22:5△~(4,7,10,13,16)n-6) methyl ester standard was detected in the transgenic yeast YFAD4 in whose culture there had been Adrenic acid Adrenic acid (ADA, C22:4△~(7,10,13,16)n-6), and the delta-4 fatty acid desaturase activity was indicated.This is the first report about the delta-4 fatty acid isolated from the marine fungi Thraustochytrium sp. FJN10…
Key words: delta-4 fatty acid desaturase; Docosahexaenoic acid (DHA); cloning and expression; Marine fungi; RACE

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