少根根霉△^12-脂肪酸脱氢酶基因在毕赤酵母中的表达

Expression of the Δ^(12)-Fatty Acid Desatuase Gene from Rhizopus Arrhizus in Pichia pastoris

为探讨采用转基因的方法将毕赤酵母改造为产多不饱和脂肪酸菌株的可行性,将来自于丝状真菌少根根霉的△^(12)-脂肪酸脱氢酶基因与毕赤酵母的胞内表达载体 pPIC3.5K 连接构建了重组表达载体 pPI-CRAD12,经 Sac Ⅰ线性化后电击转化毕赤酵母菌株 GS115获得转基因酵母菌株 PICRAD12.PCR 鉴定结果表明,目的基因已经整合到毕赤酵母基因组中.经甲醇诱导表达后,通过气相色谱和气相色谱和质谱连用的方法分析转基因毕赤酵母的脂肪酸成分表明,转基因毕赤酵母的亚油酸含量增加了4.5%,说明了可以通过增加△^(12)-脂肪酸脱氢酶基因的拷贝数或者使用强启动子的方法来增加亚油酸的含量,为将毕赤酵母改造为产多不饱和脂肪酸的基因工程菌株进行了有意义的探索.

In order to test the possibility of reconstructing the Pichia pastoris strain GS115 into a PUFAs-producing strain by genetic engineering, △^12-fatty acid desaturase gene from Rhizopus arrhizus was subcloned into the i.ntracellar expression vector pPIC3.5K to reconstruct it into a recombinant vector pPICRAD12. After linearization with Sac I , pPICRAD12 was transformed into recipient strain GS115, PCR was used to verify the successful integration into the genome of Pichia pastoris. After induction with methanol, gas chromatography was used to analyze the fatty acid component of the transgenic yeast. The result showed that linoleic acid in transgenic yeast increased 4.5 percent which indicated that it is possible to increase the content of the linoleic acid by increasing copy of the gene or by using strong promoter.