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目的构建新生隐球菌的多聚泛素UBI4低表达菌株,检测UBI4低表达对新生隐球菌生长和毒力的功能影响。方法采用融合PCR方法,扩增含有UBI4基因的片段,将其与含有组蛋白启动子的质粒融合。通过基因枪将重组质粒片段转化入新生隐球菌,应用PCR筛选、DNA序列测序以及实时定量PCR方法对基因重建株进行鉴定与验证。检测多聚泛素UBI4低表达菌株对多种应激耐受力、生长曲线以及对蜡蛾感染的毒力影响。结果成功构建了新生隐球菌多聚泛素UBI4低表达。表型检测结果提示:较之标准株,UBI4低表达株生长速率下降,对高温、H2O2和NO应激以及多种抗真菌药物敏感性显著增强;同时,UBI4下调表达后隐球菌对蜡蛾的毒力显著下调。结论我们的研究初步证实多聚泛素蛋白UBI4对新生隐球菌的生长增殖、应激应答以及感染能力具有重要的调控作用,为后续深入探讨新生隐球菌泛素系统的功能机制奠定基础。
Objective To construct a polyunsaturated UBI4 low expression strain of Cryptococcus neoformans and investigate the effect of low expression of UBI4 on the growth and virulence of Cryptococcus neoformans. Methods A fusion PCR method was used to amplify the fragment containing UBI4 gene and fuse it with the plasmid containing the histone promoter. The recombinant plasmids were transformed into Cryptococcus neoformans by gene gun. The PCR products were screened by DNA sequencing and real-time PCR. To investigate the effects of Ubiquitin UBI4 low expression strain on various stress tolerance, growth curve and virulence of WJV infection. Results Cryptococcus neoformans UBI4 low expression was successfully constructed. The results of phenotypic test suggested that the growth rate of UBI4 low expression strain was lower than that of the standard strain, and the sensitivity to high temperature, H2O2 and NO stress and multiple antifungal agents were significantly increased. Meanwhile, UBI4 down-regulated the expression of Cryptococcus neoformans Significantly reduced virulence. Conclusion Our preliminary study confirmed that Ubiquitin UBI4 plays an important regulatory role on the growth, stress response and infection ability of Cryptococcus neoformans, which lays the foundation for further exploration of the functional mechanism of Cryptococcus neoformans ubiquitin system.