无痕基因敲除技术构建鼠伤寒沙门菌luxS基因突变株

Site-directed mutagenesis of luxS in Salmonella typhimurium using scarless deletion method

为验证一种基于定点突变的无痕基因敲除技术(Scarless deletion)在鼠伤寒沙门菌中的有效性,本研究以鼠伤寒沙门菌中的lux S基因为靶基因,利用-Red重组酶和重叠延伸PCR(SOE-PCR)引入定点突变,以AAA代替lux S基因的起始密码ATG,改变lux S基因编码序列,通过测序验证并采用2型自诱导物(AI-2)试验检测是否有信号分子产生。基因测序结果显示lux S基因的起始密码ATG被引入的AAA代替;AI-2试验未检测到突变基因菌株的AI-2产物,而对照的野生菌株有AI-2产生,表明突变基因失活,方法有效。利用无痕基因敲除技术构建突变株简单快速,并且不影响被突变基因的另一条DNA单链序列,具有广泛的应用前景。

To study the effective of a scarless deletion method in bactera, the mutant of luxS in Salmonella typhimurium was constructed by a site-directed mutagenesis of scarless deletion method based on -Red recombination and splice overlap extension PCR. The sequencing of luxS gene and AI-2 assay were used to identify the mutant. The sequencing result showed that the start codon of luxS in S.typhimurium was replaced with AAA, which resulted in the gene expression inactivated. The production of AI-2 was detected by AI-2 assay and the result showed that the mutant was unable to product this signal molecule, which demonstrated that the luxS gene in the mutant was inactivated by the mutant construction method. In conclusion, the scarless deletion method was an effective method in constructing mutants of S. typhimurium.