Construction of a high-efficient expression vector of Delta12 fatty acid desaturase in peanut and its prokaryotical expression.
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.
Yin
Dongmei
D
Department of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.
Cui
Dangqun
D
Jia
Bin
B
D010367:Arachis / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D019787:Linoleic Acid / Q000378:metabolism; D019301:Oleic Acid / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D019301:Oleic Acid; D019787:Linoleic Acid; D044943:Fatty Acid Desaturases