耻垢分枝杆菌中DNA损伤与SOS反应的实验研究

SOS response induced by DNA damage in Mycobacterium smegmatis

目的探讨基因毒性物质介导的DNA损伤引起分枝杆菌SOS反应的作用。方法选择耻垢分枝杆菌为分枝杆菌模式细菌,通过紫外线损伤试验检测耻垢分枝杆菌对紫外线的敏感性,通过2倍稀释法测定利福平和氧氟沙星的最小抑菌浓度(MIC)。分别用紫外线和次抑菌浓度(1/2MIC)抗生素处理耻垢分枝杆菌,在不同时间点收集菌液,以看家基因sigA为内参,采用实时定量PCR相对定量方法检测dnaE2、recA和recX等SOS基因表达水平的变化。结果紫外线暴露45s后耻垢分枝杆菌的存活率为50%,利福平和氧氟沙星的MIC分别是32μg/ml和1μg/ml。耻垢分枝杆菌dnaE2和recX等SOS基因表达水平在紫外线下暴露45s后即开始上升,recA的表达水平在紫外线暴露3h后显著上升。耻垢分枝杆菌dnaE2和recX基因表达水平在1/2MIC的利福平作用后1h或在1/2MIC的氧氟沙星作用0h后上升,recA的表达水平在1/2MIC的利福平或氧氟沙星作用3h时显著升高。结论紫外线、抗生素等基因毒性物质可以导致耻垢分枝杆菌DNA损伤及SOS基因表达水平上升。

Objective To investigate the induction of an SOS response in Mycobacteriumsmegmatis exposed to DNA damage. Methods M.smegmatis was chosen as a model mycobacterium.M.smegmatis was tested for DNA damage by ultraviolet（UV）radiation and antibiotics.The UV sensitivity of M.smegmatis was determined based on the effect that UV irradiation had on survival.The minimum inhibitory concentrations（MICs）of the antibiotics rifampicin and ofloxacin for M.smegmatis were determined using double dilution.After UV irradiation or treatment with a sub-inhibitory concentration（1/2 MIC）of each antibiotic,RNA was extracted from each culture at 0h,1 h,3 h,and6 h using a NucleoSpin RNA II kit.Levels of expression of the SOS genes dnaE2,recA,andrecX were quantified using relative real-time PCR at the indicated time points.The gene sigA was used as an internal control.Data were statistically analyzed using the software SPSS13.0.One-way ANOVA was used to compare results,andP〈0.05 indicated a significant difference. Results M.smegmatiswas sensitive to UV irradiation.Exposure to UV radiation for 30 min led to complete cell death of M.smegmatiswhile exposure for 45 sresulted in 50%survival.After M.smegmatiswas exposed to UV radiation for 45 s,expression of the genes dnaE2（F=367,P〈0.05）and recX（F=605,P〈0.05）increased from the levels at 0h,with levels of both genes peaking at 1h.Expression of recAincreased significantly 3hafter exposure to UV radiation for 45s（F=440,P〈0.05）.The MICs of rifampicin and ofloxacin were 32μg/ml and 1μg/ml,respectively.When M.smegmatiswas treated with rifampicin at 1/2its MIC,expression of the gene dnaE2 increased from its level at 1hand peaked at 3h（F=95,P〈0.05）.Expression of recXincreased from its level at 0hand peaked at 3h（F=177,P〈0.05）.Expression of recAincreased from its level at 0hand was significantly higher at 3h（F=237,P〈0.05）.When M.smegmatiswas treated with ofloxacin at 1/2its MIC,expression of dnaE2（F=144,P〈0.05）,recA（F=106,P〈0.05）,and recX（F=65,P〈0.05）increased from levels at 0h;levels of dnaE2 and recX peaked at 3h. Conclusion DNA damage by exposure to UV radiation and antibiotics induced upregulation of SOS gene expression in M.smegmatis.