粘红酵母△^(12)-脂肪酸脱氢酶基因的克隆和表达  

Cloning and heterologous expression of a Δ^(12)-desaturase gene from Rhodotorula glutinis

作　　者：杨晓霞[1] 何仕武[1] 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,昆明650500

出　　处：《应用与环境生物学报》2015年第3期421-426,共6页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(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(KKQA201226003)资助~~

摘　　要：Δ12-脂肪酸脱氢酶一般特异性催化在油酸的Δ12位引入双键转变成亚油酸.为了从粘红酵母YM25079中克隆全长Δ12-脂肪酸脱氢酶基因序列,参考已知的Δ12-脂肪酸脱氢酶基因序列设计基因特异性引物,通过PCR扩增获得到全长为1 353 bp的cDNA序列,序列分析结果表明该序列具有一个编码450个氨基酸的完整开放阅读框,所编码蛋白质的大小为50.9×103.与报道的Δ12-脂肪酸脱氢酶一样,推测的氨基酸序列具有膜整合脂肪酸脱氢酶特异性的3个组氨酸保守区,表明该序列为一个新的编码Δ12-脂肪酸脱氢酶的基因.为了验证其功能,把开放阅读框序列亚克隆到表达载体pYES3/CT,构建重组表达载体pYRGD12,并转化到酿酒酵母的缺陷型菌株INVScl进行表达.脂肪酸气相色谱(GC)分析表明,该序列所编码的蛋白质具有Δ12-脂肪酸脱氢酶活性,能将油酸转化为亚油酸,亚油酸的含量占酵母总脂肪酸的4.31%.以上结果表明,PCR所获得序列是新的Δ12-脂肪酸脱氢酶基因.Δ^12- fatty acid desaturase generally introduces a new double bond specifically at Δ^12 position of 18C oleic acid to generate linoleic acid. This study aimed to clone the full-length Δ^12- fatty acid desaturase gene from Rhodotorula glutinis strain YM25079. Based on the sequence information of the known yeast An-fatty acid desaturase genes, a pair of specific primers was designed and used for the amplification of YM25079 Δ^12- fatty acid desaturase gene. The obtained sequence was further transformed into Saccharomyces cerevisiae strain INVScl for functional analysis. A full-length cDNA sequence of 1 353 bp was amplified from R. glutinis YM25079. Sequence analysis showed this sequence comprised an open reading frame encoding 458 amino acids of 50.9 ×10^3. The deduced amino-acid sequence showed similarity to the known Δ^12-fatty acid desaturases which comprise three characteristic conserved histidine-rich regions for the membrane-bound desaturases, indicating this cDNA sequence is a novel Δ^12-fatty acid desaturase gene. The sequence was further cloned into the expression vector pYES3/CT to generate a recombinant plasmid pY3RGD12, which was subsequently transformed into S. cerevisiae strain INVScl for heterologous expression. Total fatty acids analysis of the transformed yeast cells by gas chromatography （GC） showed a novel peak corresponding to the standards of linoleic acid methyl ester with the same retention time, which was absent in the cell transformed with empty vector. The ratio of this new fatty acid to total fatty acids was 4.31%. The results indicated that the PCR-amplified sequence is a novel Δ^12-fatty acid desaturase gene.

关 键 词：粘红酵母 Δ12-脂肪酸脱氢酶 亚油酸 克隆和表达 

分 类 号：Q78[生物学—分子生物学] Q936