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目的克隆变异链球菌ldh基因启动子,并验证其活性。方法以变异链球菌UA159基因组为模版,PCR扩增变异链球菌ldh基因候选启动子,将其插入β-葡萄糖醛酸酶(β-glucuronidase,gusA)报告基因表达载体pIB107 BamH I/XhoI之间,构建ldh基因启动子gusA报告载体pCKS11,PCR、酶切及测序鉴定;经ScaI酶线性化后转化变异链球菌UA159,卡那霉素筛选阳性克隆SCKS11,经PCR和测序鉴定后,检测其GusA活性。结果成功扩增出大小为269bp的ldh基因候选启动子;经PCR、酶切及测序鉴定,变异链球菌ldh基因候选启动子gusA报告基因表达载体pCKS11构建正确;PCR和测序鉴定,ldh基因候选启动子gusA报告株SCKS11构建成功;变异链球菌ldh基因候选启动子启动的GusA活性是无启动子的阴性对照的5.8倍,是阳性对照变异链球菌clpP基因启动子的0.9倍。结论成功克隆变异链球菌ldh基因启动子序列,具有较强的启动转录活性,为研究ldh基因的表达调控机制奠定基础。
Objective To clone the promoter of Streptococcus mutans ldh gene and verify its activity. Methods Streptococcus mutans UA159 genome was used as a template to amplify the ldh gene candidate promoter of Streptococcus mutans and inserted into the expression vector pIB107 BamH I / XhoI of β-glucuronidase (gusA) Construction of ldh promoter gusA reporter vector pCKS11, PCR, digestion and sequencing identification; ScaI enzyme linearized transformed S. mutans UA159, kanamycin screened positive clones SCKS11, identified by PCR and sequencing to detect GusA activity . Results The ldh gene candidate promoter was successfully amplified. The promoter of ldh gene promoter of S. mutans was constructed correctly by PCR, restriction endonuclease and sequencing. The PCR product was identified by PCR and sequencing, and the ldh gene candidate promoter The sub-gusA reporter strain SCKS11 was constructed successfully; the GusA activity of the S. mutans ldh gene candidate promoter was 5.8 times that of the promoterless negative control and 0.9 times that of the positive control S. mutans clpP gene promoter. Conclusion Cloning of the promoter sequence of ldh gene of Streptococcus mutans has a strong promoter activity, which lays the foundation for studying the regulation mechanism of ldh gene expression.