Article ID Journal Published Year Pages File Type
2079075 Chinese Journal of Biotechnology 2006 6 Pages PDF
Abstract
α-linolenic acid (ALA, C18:3Δ9, 12, 15) is an essential fatty acid which has many sanitary functions to human beings. However, its contents in diets are often not enough. In plants, ω-3 fatty acid desaturases (FAD) catalyze linoleic acid (LA, C18:2Δ9, 12) into ALA. The seed oil of Glycine max contains high levels of ALA. To investigate the functions of Glycine max ω-3FAD, the cDNA of GmFAD3C was amplified by RT-PCR from immature seeds, and then cloned into the shuttle expression vector p416 to generate the recombinant vector p4GFAD3C. The resulting vector was transformed into Saccharomyces cerevisiae K601 through the LiAc method. The positive clones were screened on the CM (Ura-) medium and identified by PCR, and then cultured in CM (Ura-) liquid medium with exogenous LA in 20 °C for three days. The intracellular fatty acid composition of the engineering strain Kp416 and Kp4GFAD3C was analyzed by gas chromatography (GC). A novel peak in the strain Kp4GFAD3C was detected, which was not detectable in control. Comparison of the retention times of the newly yielded peak with that of authentic standard indicated that the fatty acid was ALA. The content of ALA reached 3.1 % of the total fatty acid in a recombinant strain, the content of LA correspondingly decreased from 22 % to 16.2 % by contrast. It was suggested that the protein encoded by GmFAD3C can specifically catalyze 18 carbon PUFA substrate of LA into ALA by taking off hydrogen atoms at Δ15location. In this study, we expressed a Glycine max ω-3 fatty acid desaturase gene in S. cerevisiae; an efficient and economical yeast expressing system (K601-p416 system) which is suitable for the expression of FAD was built.
Related Topics
Life Sciences Biochemistry, Genetics and Molecular Biology Biotechnology
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