缺刻缘绿藻ω3脂肪酸去饱和酶基因(ω3FAD)在酿酒酵母中的低温诱导表达

摘要

ω3脂肪酸去饱和酶(fatty acid desaturase,FAD)能使藻类细胞产生一系列具有高附加值的ω3脂肪酸.在已克隆到缺刻缘绿藻(Myrmecia incisa Reisigl)的ω3FAD基因基础上,为进一步了解其功能,本研究首先利用反转录PCR(RT-PCR)技术,克隆其开放阅读框(open reading frame,ORF)片段,然后亚克隆到穿梭表达载体pYES2中,以构建重组酵母表达载体pY-ω3FAD;通过电穿孔法将该重组载体转入酿酒酵母(Saccharomyces cerevisiae)INVSc1菌株中,经筛选与序列验证得到含有pY-ω3FAD重组质粒的酵母转化株.在5℃,添加底物亚麻酸及半乳糖诱导表达,连续培养72 h,经脂肪酸甲酯的气相色谱分析及气相色谱-质谱联用证明,转目的基因的酵母能将外源添加的亚油酸在15位脱氢生成α-亚麻酸,表明ω3FAD具有△15脂肪酸去饱和作用的功能.在不同温度条件下诱导培养时,气相色谱结果显示,在30℃培养的转基因酵母中没有检测到α-亚麻酸；但在不高于25℃培养的转基因酵母中,发现ω3FAD能将外源添加的底物去饱和为α-亚麻酸,且随着温度的降低,其去饱和能力增强,5℃时的底物转化效率达到29.73％.将转目的基因酵母在5℃温度下诱导培养不同时间,结果显示,随着培养时间的增加,LA(linoleic acid,亚油酸)转化为α-亚麻酸的效率也提高,培养4d时其转化效率达到38.86％.该研究结果提示,缺刻缘绿藻ω3FAD基因编码的酶蛋白为一个低温诱导酶.缺刻缘绿藻ω3FAD基因之所以能在酵母细胞中被低温诱导表达,可能因为后者存在一个低温诱导的脂肪酸去饱和系统.%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 INVScl 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 a-linolenic acid at the A15 position of carbon chain. This result suggested that the ω3FAD cloned from M. Incisa at least functioned as a A15 FAD for the conversion of linoleic acid to a-linolenic acid. GC analysis demonstrated that the new product of a-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 a-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.