费氏中华根瘤菌与耐盐有关基因的克隆及其在大肠杆菌中的表达  

Cloning of Gene Related to Salt Tolerance from Sinorhizobium fredii RT19 and Its Expression in Escherichia coli

作　　者：葛世超[1] 刘艳宁[1] 杨苏声[1] 

机构地区：[1]中国农业大学生物学院微生物学系,北京100094

出　　处：《Acta Genetica Sinica》2001年第6期575-582,共8页

基　　金：国家自然科学基金(No.39270025);欧盟科研基金项目(No. IC18CT960103)资助&&

摘　　要：费氏中华根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ ｆｒｅｄｉｉ）ＲＴ１９与耐盐有关的４．４ｋｂ ＤＮＡ片段含有３个相间的开放阅读框，经亚克隆及功能检测，证实其中的ＯＲＦ２与耐盐有关。将ＯＲＦ２分别克隆到表达载体以ｐＴＨｉｏＨｉｓＡ、Ｂ和Ｃ上，得到３个重组质粒ｐＧＡ、ｐＧＢ和ｐＧＣ，转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）ＤＨ５α。经ＩＰＴＧ诱导后和作ＳＤＳ－ＰＡＧＥ分析，发现只有ｐＧＣ编码的融合蛋白获得了表达。其分子量为５３ｋＤａ，恰好是ｔｒｘＡ基因编码的硫氧还蛋白与推测的蛋白质分子量之和。Ｗｅｓｔｅｒｎ印迹证实，表达的蛋白质是ｔｒｘＡ基因和目的基因编码的。A 4.4kb DNA fragment related to salt tolerance containing three open reading frames was isolated from the gene library of S fredii strain RT19. By subcloning and functional analysis, only ORF2 related to salt tolerance was obtained. The ORF2 was ligated to expression vectors pThioHisA, B and C, respectively, and recombinant expression vectors pGA, pGB and pGC containing 1.5kb DNA fragment related to salt tolerance were constructed. These recombinant expression vectors were transformed into E. coli DH5α. Inducing by IPTG and analyzing with SDS-PAGE, it was found that the fusion protein encoded by pGC was expressed, and its molecular weight was equal to the sum of thioredoxin encoded by trxA and ORF2 putative protein molecular weight. The Western blot demonstrated that the target gene was successfully expressed in E. coli.

关 键 词：费氏中华根瘤菌 耐盐 克隆 表达 大肠杆菌 

分 类 号：Q78[生物学—分子生物学]