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| Low-temperature-induced Expression of a ω3 Fatty Acid Desaturase Gene(ω3FAD) from Myrmecia incisa in Saccharomyces cerevisiae |
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Abstract Omega 3 fatty acid desaturase (FAD) enables algae to produce a series species of ω3 fatty acids of high-valued quality. Based on the sequence of a ω3FAD gene cloned from Myrmecia incisa Reisigl., its open reading frame(ORF) was amplified by RT-PCR and sub-cloned into the shuttle vector pYES2 to generate the recombinant vector pY-ω3FAD. This recombinant plasmid was transformed into a defective mutant INVSc1 strain of Saccharomyces cerevisiae for expression by electroporation. The target gene integrated in the yeast genome was confirmed by screening and sequencing. The transgenic yeast was cultured at 5℃ for 72 h with linoleic acid as a substrate and galactose as an inducer. Gas chromatography(GC) of fatty acid methyl esters and GC-mass spectrometry analysis showed that the transgenic yeast with the recombinant vector pY-ω3FAD could catalyze linoleic acid into α-linolenic acid at the Δ15 position of carbon chain. This result suggested that the ω3FAD cloned from M. incisa at least functioned as a Δ15 FAD for the conversion of linoleic acid to α-linolenic acid. GC analysis demonstrated that the new product of α-linolenic acid could not be detected from the transgenic yeast if it was cultured at 30℃. However, the exogenous precursor linoleic acid could be converted into α-linolenic acid by the transgenic yeast with target gene while it was cultured at 25℃and lower than this temperature. As the temperature decreased, the conversion of linoleic acid by the transgenic yeast would be enhanced and it reached 29.73% at 5℃. GC analysis also indicated that the conversion of linoleic acid by the transgenic yeast would be increased when the incubation time was delayed at 5℃. The conversion reached 38.86% while the transgenic yeast was cultured for 4 d. The results suggest that the protein encoded by the ω3 FAD gene from M. incisa can be a low-temperature inducible enzyme. The successful expression of the ω3 FAD gene from M. incisa in yeast induced by low temperature may be the reason that yeast has possessed a complete adaptation system to low temperature in fatty acid desaturation
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Received: 08 February 2012
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