The Eukaryotic Expression Of Δ9 And Δ6-fatty Acid Desaturase Of Eriocheir Sinensis

Fatty acids are an important source of nutrition,polyunsaturated fatty acids(PUFAs)are playing an important role in sustaining and regulating cell normal biological functions;are also the precursor of many important bioactive molecules.Polyunsaturated fatty acid synthesis refers to saturated fatty acids as the substrate,through a series of desaturation enzyme and Elongase enzyme.Desaturation enzyme is the key enzyme of polyunsaturated fatty acid synthesis pathway,it controls the degree of unsaturated fatty acids,and its role in catalytic dehydrogenation and carrier of saturated fatty acids and unsaturated fatty acid in the double bond is formed on the acyl chains,so that the unsaturated degree increase.Δ6-Fatty acid desaturase andΔ9-Fatty acid desaturase are all the important members in the polyunsaturated fatty acid synthesis,which can catalytic fatty acid chains to form a double bond in a particular location.We designed primers based on the Δ9-FAD cDNA sequence(Accession Number:JQ693685)and Δ6-FAD sequence(Accession Number:KP876058)from Eriocheir sinensis to obtain the opening reading frame(ORF).The Δ9-FAD and Δ6-FAD eukaryotic expression are constructed to verify the genes function.Test method of SDS-PAGE 、GC-MS and Westernblot are used for analysis of the experimental results.Saccharomyces cerevisiae expression system was INVSC1,the selected expression vector was pYES2.According to the cloned Δ6 and Δ9-FAD ORF and vector pYES2 sequence,we designed primers respectively,and each pair of primers with 6xHis The target gene sequence of Δ6 and Δ9-FAD containing 6xHis tag was obtained by PCR,and the target gene and vector were digested with the fast cleavage enzyme.And then through the T4 DNA ligase to connect the target gene fragment and the vector which had been double digested to obtain recombinant plasmid.The correct recombinant plasmids were transferred into the prepared yeast competent cells by electrotransfer,and cultured on SC-U solid culture at 30 ℃ for 24-36 h.The positive strains were picked foryeast bacteria liquid PCR.The correct bacteria was send to the biological company for sequencing verification,The correct strains were induced by SC-U galactose culture at16 ℃,25 ℃ and 30 ℃ respectively.The samples were harvested at 24 h,48h,72 h and96h,then be dectected by Westernblot and GC_MS.Results indicated that recombinant Eriocheir sinensis with△6 and Δ9-FAD aren’t agree with the expected.Pichia pastoris expression system was GS115,pPIC3.5K was chose as Δ9-FAD expression vector,Δ6-FAD expression vector was pPICZαA.In the same way,the primers were designed again according to the ORF of the Δ6 and Δ9-FAD and the sequences of the corresponding vectors respectively.At the same time,the HA labels were added to each pair of primers,and the Δ6 and the HA Δ9-FAD gene sequence,respectively,with the selected fast cut enzyme,and then through the T4 DNA ligase to obtain recombinant plasmid.Transferring the recombinant plasmids into yeast cells and the positive strains were sequenced.The correct strains were induced at 30 ℃ and tested at 48 h.Results showed that recombinant P.pastoris GS115 with Δ9-FAD aren’t agree with the expected.Δ6-FAD recombinant plasmid was linearized by Sac1 and transferred into yeast.The bacterial cells were detected by WB,and the target protein bands were found at 60 KD.