高山被孢霉Δ^6-脂肪酸脱氢酶基因在红冬孢酵母中的表达  

Expression of Mortierella alpine Δ^6-Desaturase Gene in Rhodosporidium Kratochvilovae

作　　者：李凌彦[1] 胡彬彬[1] 赵吉然 魏云林[1] 林连兵[1] 季秀玲[1] 张琦[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,中国云南昆明650500

出　　处：《生命科学研究》2014年第4期304-309,共6页Life Science Research

基　　金：国家自然科学基金资助项目(31160016;31260034);云南省应用基础研究基金资助项目(KKSA201126005);教育部回国人员科研启动基金资助项目(KKQA201226003)

摘　　要：以绿色荧光蛋白(green fluorescent protein,GFP)作为报告基因,将质粒pRH2304转化红冬孢酵母YM25235进行表达分析,荧光显微观察结果表明GFP在YM25235获得表达,建立了红冬孢酵母YM25235遗传转化方法。在此基础上,以高山被孢霉Δ6-脂肪酸脱氢酶基因取代pRH2304中的GFP基因,构建重组质粒pRH2304MAD6,将其转化红冬孢酵母YM25235进行表达分析。PCR结果表明,高山被孢霉Δ6-脂肪酸脱氢酶基因已经整合到YM25235基因组中,进一步的脂肪酸气相色谱分析结果表明,该基因编码产物催化n-6途径中的亚油酸转化成γ-亚麻酸,占细胞总脂肪酸的4.35%,但没有检测到催化n-3途径中的α-亚麻酸转化成十八碳四烯酸。Using green fluorescent protein（GFP） as a reporter gene, the plasmid pRH2304 was electroporated into Rhodosporidium Kratochvilovae strain YM25235 for expression analysis. Fluorescent microscopy revealed that the GFP gene was successfully expressed in the transgenic yeast cells, which indicated that the transformation method of pRH2304 into YM25235 was established. Based on this, the GFP gene on the plasmid was replaced by Mortierella alpine Δ^6-desaturase gene to construct a new recombinant plasmid pRH2304MAD6, which was further transformed into YM25235 for expression. PCR result showed that the target gene has been integrated into the genome of YM25235. Subsequent GC analysis of fatty acids from transgenic yeasts indicated that the coding product of Mortierella alpine Δ^6-desaturase gene catalyzed the conversion of n-6 fatty acid linoleic acid into γ-linolenic acid. The percentage of γ-linolenic acid to total fatty acids was 4.35%. However, no conversion of n-3 fatty acid α-linolenic acid into steridonic acid catalyzed by the same Δ^6-desaturase was detected.

关 键 词：红冬孢酵母 高山被孢霉 Δ^6-脂肪酸脱氢酶基因 Γ-亚麻酸 表达 

分 类 号：Q786[生物学—分子生物学]