白念珠菌 HGT 19基因敲除株的构建与基因功能初步研究

Construction of an HGT 19 gene deficient Candida albicans strain and preliminary assessment of gene function

目的构建白念珠菌HGT 19基因敲除株,并进行初步的基因功能研究。方法利用同源重组的原理,采用LEU2、HIS1和ARG4营养筛选策略,选用乙酸锂法转化白念珠菌,同时替换白念珠菌原基因组的两条链,得到HGT 19敲除菌(HGT 19Δ/Δ)。利用敲除菌与野生型对照开展一系列表型研究,初步探索HGT 19在白念珠菌中的功能。结果经过PCR和营养型鉴定,成功获得HGT 19敲除株。点板实验表明HGT 19Δ/Δ与野生型相比,HGT 19Δ/Δ对Zn^(2+)与细胞膜损伤剂SDS(十二烷基硫酸钠)敏感,对其他大部分金属离子(Na^(+)、K^(+)、Li^(+)、Ca^(2+)、Fe^(2+)、Fe^(3+)、Cu^(2+))无明显差异。生物膜实验表明HGT 19Δ/Δ生物膜与野生型相比有缺陷。小鼠的生存实验表明,HGT 19Δ/Δ组与野生型组相比,可减缓小鼠的死亡。结论敲除HGT 19基因后,白念珠菌对锌离子敏感,形成生物膜能力明显减弱,毒力明显减弱。

This study was aimed at constructing an HGT 19 gene knockout strain of Candida albicans and conducting preliminary gene function studies.Based on homologous recombination,LEU2,HIS1 and ARG4 nutritional screening strategies,and the lithium acetate method were used to transform Candida albicans.The two chains of Candida albicans genogenic genome were replaced to obtain the HGT 19 knockout strain(HGT 19Δ/Δ).To preliminarily explore the function of HGT 19 in Candida albicans,we performed a series of phenotypic studies using the knockout strain and a wild type control.The HGT 19 knockout strain was successfully obtained,based on PCR and vegetative type identification.Compared with wild type,HGT 19Δ/Δwas sensitive to Zn^(2+)and Sodium dodecyl sulfate,but not to most other metal ions(Na^(+),K,Li^(+),Ca^(2+),Fe^(2+),Fe^(3+),Cu^(2+)).Biofilm experiments showed that HGT 19Δ/Δbiofilms were defective,in contrast to wild type biofilms.Survival experiments in mice demonstrated that the HGT 19Δ/Δgroup,compared with the wild type group,showed slowed death.Compared with the wild type,the HGT 19 gene knockout strain was sensitive to zinc ions,and its biofilm formation ability and virulence were significantly weaker.