变异链球菌ldh基因启动子的克隆及活性检测

Activity detection of ldh gene promoter of Streptococcus mutans

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。

We cloned the ldh gene promoter of Streptococcus mutans and explored it′s activity.The candidate promoter of S.mutans ldhgene was amplified fromS.mutans UA159 chromosome DNA by PCR,then was inserted into pIB107 by BamH I/XhoI to constructβ-glucuronidase report plasmids pCKS11.The plasmids pCKS11 were verified by PCR,restriction enzyme and sequencing.After being linearized by ScaI,the plasmids pCKS11 were transformed into S.mutans UA159.Then,the strain was screened out by THY agar contain 300μg/mL kanamycin.After being confirmed by PCR and sequencing,the GusA activity of homologous recombinationβ-glucuronidase report strain SCKS11 and it parental strains were measured.Results showed that the 269 bp candidate promoter of ldh gene was amplified successfully.PCR,restrictive endonuclease digest and sequencing confirmed the validity of the ldh-gusA report plasmids and strains.The result of GusA assays showed that the activity of the candidate promoter of S.mutans ldh gene were 5.8folds of that of the negative control without any promoter and 0.9folds of that of the clpPgene promoter of S.mutans positive control.The success of locating the promoter of S.mutans ldh gene and construction of report strains offered an experimental basis to the further study of the expression and regulation of ldh gene of S.mutans.