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 LIU Chunyu,CHEN Yan,HUANG Jinqun,et al.Direct Construction and Screening of the Expression Vector of Anaerobic Clostridium in Escherichia coli[J].Chinese Journal of Applied & Environmental Biology,2013,19(05):822-826.[doi:10.3724/SP.J.1145.2013.00822]





Direct Construction and Screening of the Expression Vector of Anaerobic Clostridium in Escherichia coli
(1广西大学生命科学与技术学院 南宁 530005) (2广西科学院国家非粮生物质能源工程技术研究中心 南宁 530003)
LIU Chunyu CHEN Yan HUANG Jinqun PEI Jianxin PANG Hao HUANG Ribo
(1Life Science and Technology College, Guangxi University, Nanning 530005, China) (2National Engineering Research Center for Non-food Biorefinery, Guangxi Academy of Sciences, Nanning 530003, China)
厌氧梭菌硫解酶启动子大肠杆菌重组表达 纤维素酶菌株筛选
anaerobic Clostridium thiolase promoter Escherichia coli recombinant expression cellulase strain screening
Q936 : Q78
厌氧微生物的代谢途径改造、重组基因表达等分子生物学操作步骤多,易造成氧气接触,从而导致厌氧微生物死亡. 针对厌氧梭菌-大肠杆菌穿梭载体的构建和筛选问题,利用纤维素内切酶基因Cphy3367作为报告基因,分析穿梭载体的硫解酶启动子在大肠杆菌中启动基因转录表达的可能性. 含有重组质粒pSOS95-Cphy3367的大肠杆菌菌株相对于对照菌株生长迟缓,生物量降低. 实时定量PCR检测及重组蛋白酶活检测结果中,发现硫解酶启动子在大肠杆菌中能启动基因转录表达,在37 ℃温度下48 h后达到高峰. 表明硫解酶启动子在大肠杆菌中可以被宿主识别和启动基因表达,基因可以翻译成为蛋白质. 在厌氧梭菌的穿梭载体构建中,含有硫解酶启动子的载体可以在大肠杆菌中进行重组载体的活性筛选这个步骤,从而降低梭菌厌氧操作的复杂度和难度. 图4 表1 参24
The complex procedures of molecular biological experiments with anaerobic microbes, such as reconstruction of metabolic pathway and expression of the recombinant gene, would introduce oxygen easily, which may result in death of the anaerobic microorganisms. This study was aimed at the problem that the process of construction and screening of shuttle vector in Clostridium would make strains be exposed to oxygen and die. The possibility of initializing gene transcription by the thiolase promoter in Escherichia coli was analyzed using endoglucanase gene Cphy3367 as a reporter gene. Comparing with control strain, recombinant strains contained pSOS95-Cphy3367 plasmid grown more slowly and had a biomass reduction. The results of real-time quantitative PCR and activity detection of recombinant enzyme showed that the thiolase promoter in E. coli initiated the expression of gene Cphy3367. The initiation of gene Cphy3367 peaked at 48 h incubation at 37 ℃. In conclusion, the thiolase promoter can be recognized by host cell and the gene can be translated into protein in E. coli. The anaerobic Clostridium shuttle vector containing the thiolase promoter can be actively screened in E. coli, thereby reduce complexity and difficulty of the anaerobic operation. Fig 4, Tab 1, Ref 24


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国家自然科学基金项目(21366007)和广西科学研究与技术开发项目(1347004-1)资助 Supported by the National Natural Science Foundation of China (No. 21366007), and the Guangxi Program for Science and Technology Development (No. 1347004-1)
更新日期/Last Update: 2013-10-28