[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 hispa...[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.展开更多
文摘[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.
