Expression Of Delta6Fatty Acid Desaturase From Mortierella Isabellina In Escherichia Coli And Yeast

Arachidonic acid is an important unsaturated fatty acid, which is an essential unsaturated fatty acid of thehuman body. But it can not synthesised in the human body. Relative to the traditional biological extraction, theemerging microbial fermentation method has many advantages. Mortierella is one of the important mold ofthem. Deep research on Mortierella metabolic process of arachidonic acid△6fatty acid desaturase isindispensable for a rate-limiting enzyme, therefore, the study of△6fatty acid desaturase regulation in themetabolic process is of great significance.The primers were designed according to the sequences of△6fatty acid desaturase gene in the GenBankand the multipleclone sites of the sequence of pET-32a(+)vector.Extract the genome RNA of Lactobacillusplantarum and amplify△6fatty acid desaturase gene from Lactobacillus by PCR and clone into expressionvector pMD18-T,transform the constructed recombinant plasmid to E.coli DH5α,through PCR,doubledigesting,T4-ligase Iigating and potsereening,the fragment of△6fatty acid desaturase gene was subclonedinto the pET32a(+).The idenitified plasmid was transferred into E.coli BL21(DE3)under induction ofIPTG.Purifed by the expressed product by electroeluting and identified by SDS-PAGE and Western bloting,the△6fatty acid desaturase gene was amplified with the template by PCR.The Primers were designed withmultiple clone sites in the expression vector pGAPZαA and the△6fatty acid desaturase gene,the exactlyidentified plasmid was digested with EcoRⅠand XhoⅠ,then ligated with the digested pGAPZαA.The△6fatty acid desaturase fragment was subcloned into the pGAPZαA.Then the plasmid was transfected inio E.coliDH5α.The results of PCR and enzyme digestion of plasmid proved that recombination vector wasobtained.After ligation of the plasmids by AvrⅡ, being integrated into the genome of host yeast P.pastorisSMD1168by electroporation and sereening the converter by ZeocinTM,the results of PCR of plasmid provedthat recombination vector was obtained, and the Immunologic competence was detected with SDS-PAGE andWestern bloting.In this research,the△6fatty acid desaturase gene from Mortierella was cloned.The construeted plasmidwas construeted suceessfully,SDS-PAGE analyzing indicated a molecular weight of70kDa(with6histag)protein was obtained at inducing3hours under induction of1.0mmoL/L IPTG.The fused Protein wasidentified by westem blot with antigenicity.In addition, recombination plasmid was construeted as well and the△6fatty acid desaturase gene was successfully expressed in P.pastoris SMD1168.SDS-PAGE and Westernbloting analysis showed that a high-level expression of65kDa of architectural gene of△6fatty acid desaturasewith immunologic competence,higher purity of the purified protein can be used for follow-up test.