编码S-腺苷-L-高半胱氨酸水解酶的草菇冷诱导基因Cor3功能验证  

Cold-Induced Volvariella volvacea Cor3 Gene Encodes an S-Adenosyl-L-homocysteine Hydrolase

作　　者：孙晓红[1,2,3] 汪虹[1] 杨晓达[4] 冯爱萍[1] 陈明杰[1] 潘迎捷[3] 

机构地区：[1]上海市农业科学院食用菌研究所,农业部南方食用菌资源利用重点实验室,国家食用菌工程技术研究中心,国家食用菌加工技术研发分中心,上海市农业遗传育种重点开放实验室,上海201403 [2]南京农业大学生命技术学院,江苏南京210095 [3]上海海洋大学,上海201306 [4]北京大学医学部药学院,北京100083

出　　处：《食用菌学报》2012年第1期13-16,共4页Acta Edulis Fungi

基　　金：草菇基因组和功能基因组数据库共享平台建设(编号:10dz2212400);上海市科学技术委员会重点项目的部分研究内容

摘　　要：将草菇(Volvariella volvacea)冷诱导基因Cor3cDNA序列经EcoR I酶切克隆到原核表达载体pPROK-1中,转化大肠杆菌(Escherichia coli)JM109,经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达、分离纯化发现该蛋白的分子量约为43kD,等电聚焦电泳结果表明,该蛋白的等电点为6.1,HPLC检测说明合成方向的酶活性为每毫克蛋白每分钟4.5μmol,DNTB与同型半胱氨酸颜色反应测定水解方向的酶活性为每毫克蛋白每分钟0.7μmol,根据上述特性可以确定草菇冷诱导基因Cor3所编码的蛋白是S-腺苷同型半胱氨酸水解酶(SAHH)。The full-length cDNA sequence of Volvariella volvacea Cor3 gene was digested with EcoRI restriction enzyme,ligated to the expression vector pPROK-1,and the recombinant plasmid was transformed into Escherichia coli JM109.The encoded protein was over-expressed in the transformed bacteria by addition of 0.5 mmol/L IPTG to the culture medium and cell-free extracts were prepared by sonication.DEAE-cellulose ion-exchange,Sephacryl S-200 gel filtration and QAE-Sepharose negative ion exchange chromatography yielded a single protein band on SDS-PAGE with a molecular mass of 43 kDa and an isoelectric point of 6.1.The purified protein was identified as S-adenosyl-L-homocysteine hydrolase with a synthetic activity（measured by HPLC） of 4.5 μmol/min/mg protein,and a hydrolytic activity（measured by titration of the product homocysteine with 5,5＇-dithiobis（2-nitrobenzoic acid） of 0.7 μmol/min/mg.

关 键 词：S-腺苷同型半胱氨酸水解酶基因 草菇 冷诱导基因 

分 类 号：S646.13[农业科学—蔬菜学]