白念珠菌ERG3基因敲除及其对耐药性的影响

Knocking out ERG3 gene of Candida albicans and its effect on drug resistance

目的·构建白念珠菌ERG3基因敲除株,分析ERG3敲除对白念珠菌唑类药物耐药性的影响及其常见耐药基因的表达。方法·使用融合PCR和同源重组技术,以白念珠菌SN152菌株基因组DNA、带有筛选标记的质粒DNA为模板构建敲除组件。采用醋酸锂转染法将敲除组件转染入SN152中,从而获得ERG3^+/-和ERG3^-/-菌株。采用微量肉汤稀释法来判断SN152、ERG3^+/-和ERG3^-/-菌株对唑类药物的耐药性差异,同时使用实时荧光定量PCR检测菌株中的常见耐药基因。结果·获得白念珠菌ERG3敲除株。ERG3^-/-菌株对唑类药物的耐药性明显高于ERG3^+/-和SN152菌株。ERG3^-/-菌株的常见耐药基因(CDR1、CDR2、MDR1、ERG11)表达显著上升(均P<0.05)。结论·成功构建白念珠菌ERG3敲除株。ERG3基因的敲除提高了白念珠菌唑类药物的耐药性,且该基因表达下调的同时伴随其他耐药基因的表达上调。

Objective·To construct the ERG3 gene knockout strain of Candida albicans,and to analyze the effect of ERG3 knockout on the resistance of Candida albicans to azole drugs and the expression of its common resistance genes.Methods·Knockout components were constructed with genomic DNA of Candida albicans SN152 and plasmid DNA with screening markers as templates by fusion PCR and homologous recombination techniques.The knockout components were transfected into Candida albicans SN152 by lithium acetate transfection method to obtain ERG3^+/-and ERG3^-/-strains.The differences of drug resistance in SN152,ERG3^+/-and ERG3^-/-strains to azole drugs were determined by microbroth dilution method.Meanwhile,realtime quantitative PCR(qPCR)was used to detect the expression of drug resistance-related genes in the three strains.Results·Candida albicans ERG3 gene knockout strains were efficiently constructed.The resistance of ERG3^-/-strains to azole drugs was significantly higher than that of ERG3^+/-and SN152 strains.The expression of drug resistance-related genes(CDR1,CDR2,MDR1,ERG11)in ERG3^-/-strains increased significantly(all P<0.05).Conclusion·Candida albicans SN152 ERG3^-/-double allelic knockout strain is successfully constructed.The knockout of ERG3 gene improves the resistance of Candida albicans to azole drugs,and the down-regulation of ERG3 gene expression is accompanied by the up-regulation of other resistance related genes.