Construction of a High-efficient Expression Vector of A Fatty Acid Desaturase in Peanut and Its Prokaryotical Expression

摘要

A full-length sequence coding for A~12 fatty acid desaturase gene from pesamt(Arachis hypogaea L.)was cloned into the expression vector, pRSETB, to generate recombinant plasmid pRSET/HO-A, which was subsequently transformed into expression Escherichia. coli BL21(DE3)pLysS. The A12 fatty acid desaturase was highly expressed in E. coli BL21(DE3)pLysS in the presence of isopropyl-D-thiogalactopyranoside (IPTG). The fusion protein was purified and used to form a reaction system in vitro by adding oleic acid as substrate and incubating it at 20 deg C for 6 h. Total fatty acids was extracted and methlesterified and then analyzed with gas chromatography. A novel peak corresponding to linoleic acid methyl ester standards was detected with the same retentiontime. GC-MS (gas chromatogram and gas chromatogram-mass spectrometry) analysis showed that the novel peak was linoleic acid methyl ester. These results exhibited A~12 fatty acid desaturase activity, which could convert oleic acid to linoleic acid specifically.