Construction of a High-efficient Expression Vector of Δ12 Fatty Acid Desaturase in Peanut and Its Prokaryotical Expression

a.
Department of Agronomy, Henan Agricultural University, Zhengzhou 450002, China
b.
The Center of Science Laboratory, Henan Academy of Agricultural Science, Zhengzhou 450002, China

摘要

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Abstract: A full-length sequence coding for Δ¹² 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 Δ¹² 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°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 Δ¹² fatty acid desaturase activity, which could convert oleic acid to linoleic acid specifically.
- peanut /
- prokaryotical expression /
- function identification

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参考文献(34)

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