长臂同源多聚酶链反应法构建变形链球菌comD基因同源重组DNA片段

Construction of Streptococcus mutans comD gene homologous recombinant DNA fragments by long flanking homology polymerase chain reaction

目的构建变形链球菌UAl59密度感应相关的comD基因同源重组DNA片段,为利用同源重组原理构建基因功能丧失菌株做准备。方法通过NCBI基因数据库获取变形链球菌的DNA序列,利用聚合酶链反应技术分别扩增变形链球菌UA159comD基因上、下游片段及抗红霉素基因片段,再通过长臂同源多聚酶链反应将这3个片段连接起来,形成同源重组DNA片段。结果经过PCR反应和琼脂电泳分析,得到了一个碱基数为3个单片段总和的连接片段,测序结果显示连接片段为预期的comD同源重组片段。结论成功构建了变形链球菌UA159comD基因同源重组DNA片段,可直接用于细菌转化构建comD基因缺陷菌株。

Objective To construct the quorum sensing related cored gene homologous recombinant DNA frag- ments of Streptococcus mutans UA159, so as to prepare for the construction of gene function-deficient strain by homologous recombination. Method The DNA sequences of streptococcus mutans UA159 was obtained through NCBI, then the upstream and downstream of coreD as well as erythromycin-resistance （ermr） gene were amplifi respectively by polymerase chain reaction（ PCR）, and the three fragments were linked together by long flanking homology polymerase chain reaction to form a homologous recombinam DNA fragment. Result PCR and agar electrophoresis analysis showed that a junction frag- ment with the equal base number as the three single fragments altogether was successfully acquired which turned out to be the expected coreD homologous recombination fragment by sequencing. Conclusion The comD gene homologous recombi- nant DNA fragments of S. mutans UA159 is successfully constructed, which can be directlly used for bacteria transformation to construct the coreD gene deficient strain.