问号钩端螺旋体Sigma N信号系统调控靶基因鉴定

Identification of Sigma N signaling system target genes in Leptospira interrogans

目的确定致病性问号钩端螺旋体(简称钩体)LA2404基因产物为Sigma N转录因子,鉴定Sigma N信号系统参与调控的下游靶基因。方法采用生物信息学软件并根据Sigma N调控基因启动子序列特征,预测并分析钩体基因组中LA2404基因及其调控的靶基因。采用等位交换原理构建问号钩体LA2404基因敲除株(ΔLA2404)。采用实时荧光定量RT-PCR(qRT-PCR)检测ΔLA2404敲除株中候选靶基因mRNA水平变化。构建LA2404基因原核表达系统并提纯目的重组蛋白(rSigma N)。采用凝胶迁移试验(gel electrophoresis mobility shift assay,EMSA)验证Sigma N调控的靶基因。结果致病性问号钩体黄疸出血群赖型赖株有一个Sigma N编码基因以及22个含Sigma N启动子序列的靶基因。ΔLA2404敲除株有钩体典型、活泼的运动方式,外形无明显变化。ΔLA2404敲除株中LA1188、LA2306、LA3426、LA1968、LA1313、LA3806和LA0773基因mRNA水平显著下调(P<0.05),其他靶基因mRNA水平无明显变化。EMSA结果显示,rSigma N可与上述靶基因启动子序列结合。结论LA2404基因产物为Sigma N调控基因。LA1188、LA2306、LA3426、LA1968、LA1313、LA3806和LA0773的启动子序列能与转录因子Sigma N结合,上述7个基因表达受Sigma N通路调控。

Objective To confirm the Sigma N transcription factor activity of a gene product encoded by LA2404 gene of Leptospira interrogans(L.interrogans)and to identify the target genes of Sigma N signaling system.Methods L.interrogans LA2404 gene and its regulated target genes were predicted using bioinformatic analysis according to the promoter sequence signature in Sigma N-regulated genes.A LA2404 gene-knockout(ΔLA2404)strain of L.interrogans was constructed based on homologous sequence recombinant of suicide plasmid.Real-time fluorescent quantitative RT-PCR(qRT-PCR)was used to detect the changes in the expression of target genes at mRNA level in theΔLA2404 mutant.A prokaryotic expression system for LA2404 gene was established and the target recombinant protein rSigma N was extracted by Ni-NTA affinity chromatography.Gel electrophoresis mobility shift assay(EMSA)was used to screen out the target genes regulated by rSigma N.Results Pathogenic L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai carried one Sigma N gene and 22 Sigma N promoter sequence-containing target genes.Qualitative examination of theΔLA2404 mutant by microscopy revealed no defect in motility and appearance.Expression of LA1188,LA2306,LA3426,LA1968,LA1313,LA3806 and LA0773 genes at mRNA level in theΔLA2404 mutant was significantly down-regulated(P<0.05),but no significant changes in the expression of other target genes at mRNA level were detected.EMSA results confirmed that rSigma N could bind to the promotor sequences of the target genes mentioned above.Conclusions Sigma N transcription factor was encoded by LA2404 gene.LA1188,LA2306,LA3426,LA1968,LA1313,LA3806 and LA0773 genes contained Sigma N promoter sequence and the expression of them was regulated by Sigma N signaling system.