极端嗜盐古菌质粒pSCM201衍生穿梭表达载体的构建及应用  

Construction and application of a novel shuttle expression vector based on haloarchaeal plasmid pSCM201

作　　者：苗荻[1] 孙超岷[1] 向华[1] 

机构地区：[1]中国科学院微生物研究所,微生物资源前期开发国家重点实验室,北京100101

出　　处：《微生物学报》2009年第8期1040-1047,共8页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30671141);国家"863计划"(2006AA09Z401)~~

摘　　要：[Objective] pSCM201 is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea.For gene expression and function analysis of specific genes in model strain Haloarcula hispanica,a novel shuttle expression vector based on pSCM201 was developed.[Methods] By combining the minimal replicon of pSCM201,the pUC19 replicon of Escherichia coli,and two antibiotic genes,a new shuttle vector between haloarchaea and E.coli was established.Furthermore,the hsp5 core promoter,multiple clone sites and a C-terminal His·Tag sequence were added to construct the expression vector pSCM307.With the help of reporter gene bgaH,the replication ability and gene expression of pSCM307 were tested in H.hispanica AS2049 by PCR identification and β-galactosidase activity assay.[Results] pSCM307 could stably self-replicate in AS2049,and bgaH was successfully expressed under the control of hsp5 promoter.[Conclusions] A user-friendly expression vector was successfully constructed in haloarchaea.[Objective] pSCM201 is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea. For gene expression and function analysis of specific genes in model strain Haloarcula hispanica, a novel shuttle expression vector based on pSCM201 was developed. [Methods] By combining the minimal replieon of pSCM201, the pUC19 replieou of Escherichia coli, and two antibiotic genes, a new shuttle vector between haloarchaea and E. coli was established. Furthermore, the hsp5 core promoter, multiple clone sites and a C-terminal His·Tag sequence were added to construct the expression vector pSCM307. With the help of reporter gene bgaH, the replication ability and gene expression of pSCM307 were tested in H. hispanica AS2049 by PCR identification and β-galactosidase activity assay. [ Results] pSCM307 could stably selfreplicate in AS2049, and bgaH was successfully expressed under the control Of hsp5 promoter. [ Conclusions] A user-friendly expression vector was successfully constructed in haloarchaea.

关 键 词：嗜盐古菌 pSCM201 穿梭载体 表达载体 

分 类 号：Q78[生物学—分子生物学]