红冬孢酵母Δ^(12)-脂肪酸脱氢酶基因的克隆和表达  

Cloning and expression of a Δ^(12)-desaturase gene from Rhodosporidium kratochvilovae

作　　者：罗敏周 杨晓霞[1] 季秀玲[1] 林连兵[1] 魏云林[1] 张琦[1] 

机构地区：[1]昆明理工大学生命科学与技术学院生物工程研究中心,云南昆明650500

出　　处：《云南大学学报（自然科学版）》2016年第4期669-675,共7页Journal of Yunnan University(Natural Sciences Edition)

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

摘　　要：Δ^(12)-脂肪酸脱氢酶是多不饱和脂肪酸合成的关键酶.参考已知的Δ^(12)-脂肪酸脱氢酶基因序列设计特异性引物,通过PCR扩增从红冬孢酵母YM25235中获得了全长为1 341 bp的一个cDNA序列.序列分析表明,该序列具有一个编码446个氨基酸、相对分子质量为50.6 ku的完整开放阅读框,所编码的氨基酸序列与Δ^(12)-脂肪酸脱氢酶相似性最高但并不相同,而且也具有Δ^(12)-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Δ^(12)-脂肪酸脱氢酶的基因.为了验证功能,将该基因序列克隆到表达载体pYES3/CT中,构建重组质粒pY3RKD12,并转化到酿酒酵母的缺陷型菌株INVSCI中进行异源表达.通过脂肪酸气相色谱(GC)分析表明,在pY3RKD12转化的酵母细胞总脂肪酸中出现一个保留时间和亚油酸甲酯标准品相同的新峰,其含量占酵母总脂肪酸的3.76%,但在pYES3/CT转化的酵母细胞总脂肪酸中没有检测到.这些结果证明所克隆的序列是一个新的Δ^(12)-脂肪酸脱氢酶基因,其表达的蛋白质可催化油酸合成亚油酸.Δ^12-desaturase is a key enzyme in the biosynthesisi of polyunsaturated fatty acid.In this study, based on the sequence information of the known yeast Δ^12-desaturase genes, a pair of specific primers was designed and used for the amplification of Rhodosporidium kratochvilovae YM25235 Δ^12-desaturase gene.A fulllength coding sequence of 1 341 bp was amplified.Sequence analysis showed that the sequence contained a complete open reading frame encoding a protein of 446 amino acids with a molecular weight of 50.6 ku.The deduced amino acid sequence showed the highest similarity to the knownΔ^12-desaturases that characteristic of three conserved histidine-rich region,indicating that the cDNA sequence is a new Δ^12-desaturase gene.In order to verify its function, the coding sequence was further subcloned into expression vector pYES3/CT.Recombinant plasmid pY3RKD12 was constructed and then transformed into Saccharomyces cerevisiae strain INVScl for heterologous express.Total fatty acid analysis by gas chromatography （GC） showed that a novel peak corresponding to the standards of linoleic acid methyl ester was detected with the same retention time in pY3RKD12-tranformed yeast ceils ,which was absent in the cell transformed with pYES3/CT.The ratio of this new fatty acid to total fatty acids was 3.76%.All these results demonstrated that the cloned sequence whose coding product exhibited Δ^12-fatty acid desaturase activity to convert oleic acid to linoleic acid is a novel Δ^12-fatty acid desaturase gene.

关 键 词：红冬孢酵母 Δ^12-脂肪酸脱氢酶 亚油酸 克隆和表达 

分 类 号：Q933[生物学—微生物学]