粘红酵母delta1脂肪酸脱氢酶基因的克隆和表达.docVIP

粘红酵母?12-脂肪酸脱氢酶基因的克隆和表达收稿日期 Received: 收稿日期 Received: 接受日期 Accepted: 资助：国家自然科学基金项目(No.，云南省应用基础研究基金资助项目(No. KKSA201126005)、教育部回国人员科研启动基金(No. KKQA201226003)资助. Supported by the National Natural Science Foundation of China , the Application and Basic Research Fund of Yunnan Province (KKSA201126005), the Scientific Research Foundation for Returned Overseas Chinese Scholars, State Education Ministry of China. (KKQA201226003) **通讯作者 Corresponding author (E-mail: qzhang37@) 杨晓霞，何仕武，魏云林，林连兵，季秀玲，张琦** 昆明理工大学生命科学与技术学院 昆明 650500 摘 要 Δ12-脂肪酸脱氢酶一般特异性在18C油酸第12碳原子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，本研究参考已知的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1353bp的cDNA序列，序列分析结果表明该序列具有一个编码450个氨基酸的一个完整开放阅读框，所编码蛋白质的大小为50.9KD。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。通过脂肪酸气相色谱(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%。 关键词 粘红酵母；Δ12-脂肪酸脱氢酶；亚油酸；克隆和表达 CLC Q933 Cloning and heterologous expression of a Δ12-desaturase gene from Rhodotorula glutinis* YANG Xiaoxia, HE Shiwu, WEI Yunlin, LIN Lianbing, JI XiulingZHANG Qi** Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China Abstract Objectives: ?12- fatty acid desaturase generally introduces a new double bond specifically at Δ12 position of 18C oleic acid to generate linoleic acid. This study is to clone the full-length ?12- fatty acid desaturase gene from Rhodotorula glutinis strain YM25079. Methods: Based on the sequence information of the known yeast ?12- fatty acid desaturase genes, a pair of specific primers was designed and used for the amplification of YM25079 ?12- fatty acid desaturase gene. The obtained sequence was further transformed into Saccharomyces cerevisiae strain INVScl for functional analysis. Results: A full-length cDNA sequence of 1353bp was amplified from Rhodotorula glutinis YM25079. Sequence analysis showed thi