两株希木龙假丝酵母14α-去甲基化酶基因(ERG11)的克隆及其功能初步验证

Cloning and functional identification of 14α-demethylase genes (ERG11) from two different Candida haemulonii strains with degenerate PCR combined with RACE

为探讨希木龙假丝酵母(假丝酵母又称念珠菌)的耐药机制,首先克隆出两株希木龙念珠菌ERG11基因,初步验证其功能,从而为后续研究奠定基础。从美国国家生物技术信息中心(National Center of Biotechnology Information,NCBI)基因数据库中获取白念珠菌、热带念珠菌、近平滑念珠菌和光滑念珠菌Erg11蛋白的保守序列,设计简并引物,聚合酶链反应(polymerase chain reaction,PCR)扩增获得希木龙念珠菌ERG11cDNA部分片段;用快速cDNA末端扩增法(rapid amplification of cDNA ends,RACE)分别扩增其5′和3′端,获得完整的ERG11编码序列(coding sequence,CDS);将CDS克隆到pYES2表达载体中,在尿嘧啶营养缺陷型酿酒酵母中过表达ERG11;用微量液基稀释法检测转化后的酿酒酵母对氟康唑的敏感性,初步验证其功能。结果显示,简并PCR扩增获得预期708bp片段,5′RACE和3′RACE分别获得385bp和1 336bp片段,经纯化、克隆、测序、比对分析,获得两株菌的ERG11CDS;比对其编码的蛋白,与其他念珠菌的Erg11蛋白高度同源;分别检测克隆了这两株希木龙念珠菌ERG11CDS表达载体的酿酒酵母对氟康唑的敏感性,发现过表达ERG11明显降低其对氟康唑的敏感性。结果提示,简并PCR联合RACE能准确有效地克隆出希木龙念珠菌ERG11基因,用pYES2酿酒酵母表达系统能初步验证其功能。

ERG11genes from two different Candida haemulonii strains were cloned and their functions were verified for studying the mechanism of antifungal resistance.To obtain ERG11 gene,degenerate primers were designed according to the conserved sequences in Erg11 protein from the other four types of Candida spp.Partial ERG11 cDNA was amplified by degenerate polymerase chain reaction（PCR）.And 5′cDNA and 3′cDNA were amplified by rapid amplification of cDNA ends（RACE）method.The full-length ERG11 coding sequences（CDSs）were obtained after aligning and splicing.Furthermore,ERG11 CDSs were cloned into pYES2 and transformed into Saccharomyces cerevisiae（S.cerevisiae）which is auxotrophic for uracil.The susceptibility of yeasts to fluconazole was assayed following Clinical and Laboratory Standards Institute（CLSI）M27-A3.The results showed that the complete ERG11 CDSs were obtained and identified through homologous alignment of Erg11 proteins with other Candida spp.Moreover,the susceptibility of yeasts to fluconazole was obviously reduced by overexpression of Erg11 proteins in S.cerevisiae.It is suggested that ERG11 genes can be effectively cloned by degenerate PCR combined with RACE and their functions could be preliminarily verified in pYES2 yeast expression system.