变异链球菌hit基因缺陷菌株的构建

Construction of the hit⁃deficient mutant strain of Streptococcus mutans ATCC25175

目的构建hit基因缺陷型变异链球菌株并验证其细胞周期调控的功能。方法从变异链球菌株ATCC25175中提取基因组DNA,采用PCR扩增技术,将hit基因上下游片段克隆到pFW5质粒(壮观霉素抗性)上以构建重组质粒;利用该菌株天然遗传转化机制,将线性化重组质粒转入由其感受刺激多肽(competence stimulating peptide,CSP)刺激产生变异链球菌感受态中,进行同源重组;再通过壮观霉素抗性筛选,结合PCR产物电泳和Sanger法测序,筛选鉴定hit基因缺陷型变异链球菌株;最后在BHI培养基中培养,检测其生长速率。结果从ATCC25175变异链球菌基因组DNA中,克隆hit基因上游约856 bp、下游约519 bp片段到pFW5质粒的两个多克隆位点区间(MCS⁃I和MCS⁃II),经双酶切和PCR测序鉴定,得到重组质粒pFW5_hit_Up_Down。线性化该重组质粒,转入由CSP刺激产生变链感受态中进行同源重组;再通过壮观霉素BHI培养基多次抗性筛选,hit基因片段的PCR产物电泳和Sanger测序筛选出hit基因缺陷变异链球菌株,该缺陷菌株较其亲本菌株生长更快速(P<0.001)。结论获得具有遗传性状hit基因缺陷变异链球菌株,hit基因产物调控变异链球菌生长周期。

Objective To construct a hit⁃deficient mutant strain of S.mutans ATCC25175 and verify its cell cycle regulatory function.Method Genomic DNA was extracted from S.mutans ATCC25175 strains,and then the upstream and downstream DNA fragments of the hit gene were cloned into the pFW5 vector(spectinomycin resistant)to construct recombinant plasmids using PCR amplification.Third,employed by natural genetic transformation in S.mutans ATCC25175 strains,the linearized recombinant plasmids were transformed into their genetic competence,induced by the synthesized competence⁃stimulating peptide(CSP),and then,homologous recombination was utilized to produce crossover and noncrossover products.Fourth,the hit⁃deficient mutant strains of S.mutans ATCC25175 were screened through the spectinomycin⁃resistance marker and identified by the electrophoresis of PCR products and PCR Sanger sequencing.Finally,its growth rate in vegetative BHI medium was also investigated.Results The upstream(856 bp)and downstream(519 bp)DNA fragments of the hit gene from the genomic DNA materials of S.mutans ATCC25175 were cloned into two multiple cloning sites(MCS⁃I and MCS⁃II)of the pFW5 vector,respectively,and the recombinant plas⁃mid pFW5_hit_Up_Down was constructed and identified by double⁃emzyme digestion and PCR Sanger sequencing.The linearized recombinant plasmids were transformed into their genetic competence,induced by the synthetic CSP,and then,homologous recombination was utilized to produce various products.The hit⁃deficient mutant strains of S.mutans ATCC25175 were screened through the spectinomycin resistance marker and identified by the electrophoresis of PCR products and Sanger sequencing.The growth rate of the hit⁃deficient mutant strains versus their parental S.mutans ATCC25175 strains was increased greatly(P<0.001).Conclusion The hit⁃deficient mutant strains of S.mutans ATCC25175,having heritable traits,were successfully constructed,and the encoding Hit protein is growth⁃phase regu⁃lated in the cell cycle.