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； 汪虹； 杨晓达； 冯爱萍； 陈明杰； 潘迎捷；
【Author】 SUN Xiaohong1,2,3,WANG Hong1,YANG Xiaoda4,FENG Aiping1,CHEN Mingjie1,PAN Yingjie3(1Institute of Edible Fungi,Shanghai Academy of Agricultural Sciences;Key Laboratory of Edible Fungi Resources and Utilization(South),Ministry of Agriculture,P.R.China;National Engineering Research Center of Edible Fungi;National R&D Center for Edible Fungi Processing;Key Laboratory of Agricultural Genetics and Breeding of Shanghai,Shanghai 201403,China;2College of Life Sciences,Nanjing Agricultural University,Nanjing,Jiangsu 210095,China,3Shanghai Ocean University,Shanghai 201306,China; 4Department of Chemical Biology,School of Pharmaceutical Science,Peking University,Beijing 100083)
【机构】 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； 南京农业大学生命技术学院； 上海海洋大学； 北京大学医学部药学院；
【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)。
【Abstract】 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 chromatogr...
【关键词】 S-腺苷同型半胱氨酸水解酶基因； 草菇； 冷诱导基因；
【Key words】 S-adenosyl-L-homocysteine hydrolase gene； Volvariella volvacea； cold induced gene；
【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
【分类号】S646.13