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Construction of a high-efficient expression vector of Delta12 fatty acid desaturase in peanut and its prokaryotical
Dongmei Yin1, Dangqun Cui, Bin Jia
1Department of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.
Abstract:
A full-length sequence coding for Delta(12) fatty acid desaturase gene from peanut (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 Delta(12) 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 degrees 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 retention time. GC-MS (gas chromatogram and gas chromatogram-mass spectrometry) analysis showed that the novel peak was linoleic acid methyl ester. These results exhibited Delta(12) fatty acid desaturase activity, which could convert oleic acid to linoleic acid specifically.
