Expression Of △~4-desaturase Gene Of Thraustochytrium And 5' Flank Upstream Regulation Region Analysis

In order to determine the function ofΔ~4-desaturase of Thraustochytrium sp.FJN-10 (FAD4),the recombinant plasmid was constructed and transformed into Saccharomyces cerevisiae strain INVScl for expression.It was found to exhibitΔ~4-fatty acid desaturase activity in the presence of exogenous fatty acid substrate docosapentaenoic acid(100μmol/L)under introduction of GAL1 and DHA reached 41.13%of the total yeast fatty acid by GC detection.It was suggested that the protein encoded by FAD4 could specifically catalyze DPA into DHA.The fusion gene ofΔ~5-elongase gene(ELO5)and FAD4 was amplified by overlap extension PCR and sequenced.The recombinant plasmid containing target gene was constructed and transformed into Saccharomyces cerevisiae for expression by supplemented with 0.3 mmol/L Eicosapentaenoic acid.A novel peak corresponding to DHA methyl ester standard was detected with the same retention time which was absent in the cell transformed with empty vector.So to obtain the much more economical engineering strain which could produce DHA.A 5' flanking region DNA encoding FAD4 with about 1000bp was specifically amplified by LA-PCR,then sequenced and submitted into the GenBank data(GenBank accession No.EU074209).The sequence contained a novel promoter region with TATA box,CAAT box and GC box-like elements in the identical positions shared by the eucaryote promoter sequence regions in comparison with the sequences in the GenBank.The amplified sub-cloned products of 5' flanking region were ligated into the vector pGlow-TOPO which contained the green fluorescent protein(GFP)gene as the reporter gene to construct the expression plasmids.The recombinant plasmids were transformed into Escherichia.coli TOP10,respectively.Many Escherichia.coli cells with the recombinant plasmid could emit fluorescence.The result indicated that the 5' flanking region had the promoter gene segment,which could promote GFP gene to express in Escherichia.coli cells.