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Knock out FLO8 gene in Candida albicans by fusion PCR combined with homologous recombination

LI Wen-jing1,2, LIU Jin-yan2, SHI Ce1, WANG Ying1,2, ZHAO Yue1,2, XIANG Ming-jie1,2   

  1. 1.Department of Clinical Laboratory, Ruijin Hospital,Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 2.Radioimmunology and Clinical Laboratory, Luwan Branch, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine,Shanghai 200020, China
  • Online:2016-03-28 Published:2017-06-02
  • Supported by:

    Foundation of Science and Technology Commission of Shanghai Municipality, 15ZR1426900; Key Medical Disciplines Construction Program of Shanghai, ZK2012A21; Excellent Youth of Huangpu District of Shanghai, RCPY1407

Abstract:

Objective To knock out the FLO8 gene in Candida albicans using fusion PCR combined with homologous recombination and analyze drug sensitivity in FLO8 gene knockout strains preliminarily. Methods Upstream and downstream flanking sequences of FLO8 gene were fused with selection markers by fusion PCR to construct homologous knockout fragments, which were then transfected into Candida albicans SN152 by high efficient lithium acetate transfection method. Positive strains were screened on nutritional defect medium and two alleles in FLO8 gene were knocked out by performing the homologous recombination twice. Drug sensitivity was compared between strains with and without FLO8 gene by spot assay. Results The Candida albicans SN152 FLO8-/- double allelic deletion strains were successfully constructed. Drug resistance in FLO8-/- was greater than that in FLO8+/-, which was greater than that in SN152. Conclusion Fusion PCR combined with homologous recombination can rapidly and efficiently construct Candida albicans FLO8 gene deletion strains. Candida albicans FLO8 gene is associated with drug sensitivity and drug resistance increases with the decrease in copy number.

Key words: Candida albicans, FLO8 gene, gene knockout, drug resistance