莱茵衣藻酰基辅酶A合成酶cDNA克隆及其酵母表达  

cDNA Cloning and Yeast Expression of Acyl-CoA Synthetase of Chlamydomonas reinhardtii

作　　者：宋燕子[1] 贾彬[1] 林柏成[1] 胡章立[1] 黄瑛[1] 

机构地区：[1]深圳市海洋生物资源与生态环境重点实验室深圳市海洋藻类开发与应用工程实验室深圳大学生命科学学院,深圳518060

出　　处：《生物技术通报》2015年第9期119-124,共6页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31100582;31470431);中国博士后科学基金项目(2014M562199);深圳市科技计划项目(JCYJ20120613112512654)

摘　　要：旨在预测并克隆莱茵衣藻酰基辅酶A合成酶cDNA(cracs),分析其在酵母中的功能。RT-PCR克隆cracs序列,Clustal W和MEGA6.0软件分别分析其编码蛋白保守序列和进化树,表达并分析其在酵母YB525中的底物偏好性。结果表明,首次在莱茵衣藻中克隆获得一个cracs,测序表明其序列大小为2 004 bp,编码667个氨基酸,编码蛋白crACS的预测分子量为72.3k D,包含酰基辅酶A合成酶的两个保守区:AMP-binding区和FACS区。进化树比对显示,cr ACS与拟南芥的长链酰基辅酶A合成酶LACSs具有较高的同源性。酵母表达显示cracs编码蛋白能互补酵母YB525 LACS的缺陷表型,活化并优先利用C16∶1和C14∶0。莱茵衣藻cracs编码蛋白可活化外源脂肪酸,属于酰基辅酶A合成酶家族。This work aims to predict and clone cDNA of Chlamydomonas reinhardtii acyl-CoA synthetase（gene cracs）, and analyze its function in yeast Saccharomyces cerevisiae YB525. The cracs sequence was cloned by RT-PCR, its conserved sequence of encoded protein and phylogenetic tree were analyzed with ClustalW and MEGA6.0, then the substrate specificity in YB525 of expressed gene was analyzed. As the results, a cracs was cloned for the first time with sequence of 2 004 bp and encoded a 72.3 kD protein crACS of 667 amino acids containing two conserved regions of including acyl-CoA synthetase：the AMP-binding domain and the FACS motif. The phylogenetic tree analysis indicated that crACS shared high homology with LACs of Arabidopsis thaliana. Yeast expression experiments showed that crACS restored acyl-CoA synthetase deficient phenotype of YB525 and assimilated foreign palmitoleic acid and myristic acid. Conclusively, cracs of C. reinhardtii can activate exogenous fatty acid and belongs to acyl-CoA synthetase family.

关 键 词：莱茵衣藻 酰基辅酶A合成酶 酵母YB525 进化树 脂肪酸 

分 类 号：Q943.2[生物学—植物学]