电转化法构建变形链球菌UA159 comD缺陷菌株

Construction of comD gene mutant of Streptococcus mutans by electrotransformation

目的构建变形链球菌UAl59密度感应相关的comD基因缺陷菌株,为进一步研究该基因功能做准备。方法根据同源重组原理,利用变形链球菌UAl59comD基因同源重组DNA片段,采用电击转化方法获取转化菌落,通过形态学观察、生化特性检测、PCR及测序、RT-PCR对缺陷菌进行鉴定。结果在含有红霉素(10μg/mL)的琼脂平板上出现转化菌落,鉴定结果均显示转化菌为comD缺陷菌。结论成功构建了变形链球菌UA159comD基因缺陷菌株。

Objective To construct the quorum sensing related comD gene mutant of Streptococcus mutans UA159,and prepare for further investigation of the gene′s function.Method According to the principle of homologous recombination,the comD gene homologous recombinant DNA fragments of S.mutans UA159 was utilized to acquire transformed colony by electrotransformation,then the transformants were identified by morphological observation,biochemical characteristics testing,polymerase chain reaction（PCR） and sequencing,reverse transcription PCR,respectively.Result The transformed colonies were screened on BHI-erythromycin（10 μg/mL） agar plates,which were successfully identified as comD mutant by various analysis.Conclusion The comD gene mutant of S.mutans UA159 was constructed successfully.