DHA合成途径中关键酶基因在毕赤酵母中的表达研究

摘要

DHA ( docosahexaenoic acid) is a polyunsaturated fatty acid and essential for human being. Fishes and microalgae from marine are the main source of DHA product for now. DHA can’t be synthetized by itself in higher plant, so it will be significant to create DHA crops by genetic engineering. In this study, five genes from marine microlgae which involved in DHA synthesis were cloned, including three fatty acid desaturase genes ( D4, D5 and D6 genes encode Δ4, Δ5 and Δ6 fatty acid desaturase, respectively) and two elongase ( E18 and E20 encode C18 and C20 fatty acid elongase, respectively) . To confirm whether these optimized genes could express exactly in eukaryotes, the five target genes were cloned into pPIC9K and the resulting vectors were transformed into Pichia pastoris GS115 ( His4+Muts) . The results demonstrated the maximum proteins were obtained at 96 hours induced by methanol. Western Blot result showed that the expression product was target protein. These results laid the foundation for maize breeding containing DHA.%DHA(二十二碳六烯酸)是人体所需的一种非常重要的多不饱和脂肪酸，主要存在于深海鱼类和海洋微藻类生物中。由于高等植物自身不能合成DHA，因此通过基因工程方法在作物(如玉米)中表达DHA，将会成为最健康的DHA来源，同时也能够减轻人类对海洋资源的破坏。从深海藻类中挑选了5个DHA合成途径中的关键酶基因，分别是Δ4脱饱和酶基因( D4)、Δ5脱饱和酶基因( D5)、Δ6脱饱和酶基因( D6)、C18延长酶基因( E18)和C20延长酶基因( E20)。密码子优化并合成这5个基因，为确定优化后的核苷酸序列是否能在真核生物中正确表达，以pPIC9K为基础载体连入目的基因转化到毕赤酵母GS115( His4＋MUts)中进行表达分析。结果表明，这些基因在甲醇诱导96 h后蛋白表达量最大， Western Blot结果表明表达产物为目的蛋白。该结果为进一步培育能够自身合成DHA的作物奠定了基础。