盐生盐杆菌RM07 DNA片段在大肠杆菌中的定点诱变和启动子功能分析  

Site-directed Mutagenesis and Promoter Functional Analysis of RM07 DNA Fragment from Halobacterium halobium in Escherichia coli

作　　者：杨洋[1] 沈萍[1] 

机构地区：[1]武汉大学生命科学学院生物技术系,武汉430072

出　　处：《Acta Genetica Sinica》2004年第5期525-532,共8页

基　　金：国家自然科学基金 (编号 :3 9770 0 0 9和 2 9973 0 3 0 )~~

摘　　要：将来源于嗜盐古生菌———盐生盐杆菌 (Halobacteriumhalobium)基因组的RM0 7DNA片段以正反两个方向分别插入大肠杆菌启动子探针载体pKK2 32 8携带的报告基因———氯霉素抗性基因 (cat)的上游 ,得到RM0 7 cat融合的质粒pRM0 7 1(+)和pRM0 7 1(- ) ,将其分别转入大肠杆菌HB10 1,进而检测了不同转化子菌株的氯霉素抗性水平和细胞内氯霉素乙酰转移酶蛋白质浓度。结果表明 :正向的RM0 7片段在真细菌 (大肠杆菌 )中具有启动子活性 ,能够驱动cat报告基因的表达 ;而反向的RM0 7片段在大肠杆菌中不具有启动子活性。对RM0 7片段进行了定点诱变分析 ,检测了特定核苷酸突变对启动子活性的影响 ,结果进一步精确定位了RM0 7片段中对在大肠杆菌中的启动子功能有重要作用的关键碱基 ,并且通过改造RM0 7片段的碱基组成成分大幅提高了其在大肠杆菌中的启动子活性。The RM07 DNA fragment from Halobacterium halobium was inserted upstream of chloramphenicol acetyltransferase (cat) reporter gene in pKK232-8 in two different orientations (positively or negatively),generating the RM07-cat fusion plasmids pRM07-1(+) and pRM07-1(-).These two plasmids and pKK232-8 were transformed into Escherichia coli HB101 respectively,then the antibiotic resistance level and the chloramphenicol acetyltransferase protein concentration of different transformants were detected.The research results revealed that HB101/pRM07-1(+) was resistant to chloramphenicol and could grow on the plate containing chloramphenicol,but HB101/pRM07-1(-) and HB101/pKK232-8 were sensitive to it.Therefore,the results suggested that the positive RM07 fragment had promoter activity in Escherichia coli while the negative RM07 fragment did not.Site-directed mutagenesis of RM07 was performed by PCR mutagenesis method.The effect of specific nucleotide mutations on the chloramphenicol resistance level of different transformants was detected.The different transformants containing different nucleotide mutations were inoculated on the plates containing various concentrations of chloramphenicol and then incubated at the same time.The change of chloramphenicol resistance level reflected the change of promoter activity.By using this method,the important nucleotide responsible for the promoter function of RM07 in Escherichia coli was determined.The promoter activity of RM07 in Escherichia coli was also improved greatly by modifying the nucleotide component.

关 键 词：盐生盐杆菌 大肠杆菌 启动子功能 

分 类 号：Q933[生物学—微生物学]