Objective: To construct srtA-gene deletion mutant of Streptococcus mutans (S. mutans) UA159 with IFDC2 cassette through overlapping polymerase chain reaction (PCR) and allelic homologous recombination.
Methods: First, the upstream and downstream fragments surrounding the srtA and IFDC2 cassette were PCR amplified and ligated through overlapping PCR. The resulting amplicon was transformed into UA159, and positive transformants were selected on BHI plates containing erythromycin. Second, upstream and downstream fragments of srtA with overlap regions were generated by PCR and were overlapped to create upΔ-down amplicon. Then, the upΔ-down amplicon was transformed into the aforementioned positive transformants and selected on BHI plates containing p-Cl-Phe.
Results: The PCR analysis and DNA sequencing results indicated that the coding region of the srtA was completely deleted, and the upstream and downstream regions flanking the srtA were ligated seamlessly.
Conclusions: The markerless srtA-deletion mutant of S. mutans was constructed successfully, which laid a foundation for further study of its biological function and influence on the biofilm formation of S. mutans.
目的 利用框内缺失法，通过IFDC2基因盒及融合聚合酶链反应（PCR）、同源重组技术构建变异链球菌UA159菌株srtA基因缺失株。方法 PCR扩增变异链球菌UA159菌株srtA基因上下游同源片段及IFDC2基因盒，将这些片段连接、转化入UA159，替换srtA基因同源片段，筛选、鉴定红霉素抗性克隆；PCR扩增、连接UA159 srtA基因上下游同源片段，将连接片段转化入前述红霉素抗性克隆中替换IFDC2同源片段，筛选、鉴定抗苯丙氨酸类似物p-chloro-phenylalanine（p-Cl-Phe）克隆。结果 PCR、琼脂糖凝胶电泳及DNA测序结果均证明srtA基因编码区被完全删除，上下游片段无缝连接，成功构建UA159 srtA基因缺失株。结论 本研究成功构建了无标记的变异链球菌UA159 srtA基因缺失株，为进一步研究该基因在生物膜中的作用及其调控机制奠定了基础。.
Keywords: Streptococcus mutans; in-frame deletion system; srtA-deletion mutant.