白念珠菌CaPHM7基因的功能研究

发布时间：2018-03-29 04:03

本文选题：白念珠菌　切入点：CaPhm7　出处：《江南大学》2017年硕士论文

【摘要】：白念珠菌(Candida albicans)是常见的条件型病原真菌,共生于健康人体中,而在免疫力缺陷的病人中则通过酵母-菌丝形态的转换导致相关疾病。抗真菌药物的长期使用导致高度耐药的白念珠菌突变株大量出现,因此迫切需要开发新型抗真菌药物。钙离子稳态与白念珠菌酵母-菌丝形态的转换和致病性密切相关,因此钙离子稳态的研究可以为抗真菌药物的研发提供新的理论依据。拟南芥(Arabidopsis thaliala)AtCsc1是一个最近鉴定的钙离子通透性压力门控阳离子通道蛋白,介导高渗压力下钙离子、钠离子和钾离子的运输。我们通过氨基酸序列比对发现白念珠菌中的CaPhm7是AtCsc1的同源蛋白,但是它的功能目前仍不清楚。因此本文主要通过基因敲除等手段探究了CaPHM7基因的缺失对白念珠菌应答药物压力和菌丝发育的影响,进而推测该蛋白的功能。通过本文的研究,我们得出以下结论:1)固体平板表型分析的结果表明CaPHM7基因的缺失影响带有CaURA3基因的白念珠菌在固体平板上的生长,说明该基因可能与CaURA3基因存在遗传互作。在添加药物的YPD平板上,CaPHM7基因的杂合子缺失株对酮康唑敏感,纯合子缺失株的敏感性进一步增强,表明白念珠菌对酮康唑的敏感性与CaPHM7基因的剂量有关。此外,CaPHM7基因的缺失还导致白念珠菌对表面活性剂SDS敏感,对免疫抑制剂雷帕霉素耐受。2)菌丝发育分析的结果表明CaPHM7基因的缺失导致白念珠菌在液体YPD+10%胎牛血清(Fetal Bovine Serum,FBS)、Spider和Lee’s培养基中菌丝发育的缺陷,以及在液体Spider培养基中细胞聚集和絮凝的缺陷。此外,CaPHM7基因的缺失还导致白念珠菌在固体Spider和YPD+10%FBS平板上菌落生长的缺陷,以及在固体Spider和Lee’s平板上菌丝发育的缺陷。3)GFP荧光定位分析的结果表明CaPhm7存在于白念珠菌酵母态和菌丝态细胞的质膜上,而SDS、雷帕霉素和酮康唑的处理不影响该蛋白的亚细胞定位。4)同源蛋白功能互补分析的结果表明CaPHM7不能互补同源基因ScPHM7在酿酒酵母(Saccharomyces cerevisiae)细胞应对雷帕霉素、氯化锌和衣霉素压力时的功能。5)CaSpo75是AtCsc1在白念珠菌中的另一个同源蛋白,本文研究发现CaSPO75基因可能具有负向调控细胞壁压力和特比萘芬压力,正向调控酮康唑压力以及参与白念珠菌胞内锌离子和锰离子稳态调控的功能。
[Abstract]:Candida albicansis, a common opportunistic pathogen, is symbiotic in healthy people. In immunodeficient patients, however, yeast-mycelium morphologies lead to related diseases. The long-term use of antifungal drugs leads to the emergence of highly resistant mutant strains of Candida albicans. Therefore, there is an urgent need to develop new antifungal drugs. Calcium homeostasis is closely related to the morphological transformation and pathogenicity of Candida albicans yeast and hyphae. Therefore, the study of calcium homeostasis may provide a new theoretical basis for the development of antifungal drugs. Arabidopsis thaliala)AtCsc1 is a recently identified calcium ion permeability pressure gated cationic channel protein, which mediates calcium ion under hyperosmotic pressure. We found that CaPhm7 in Candida albicans is the homologous protein of AtCsc1 by amino acid sequence alignment. However, its function is still unclear. Therefore, the effects of CaPHM7 gene deletion on drug stress and hyphal development of Candida albicans were investigated by gene knockout, and the function of the protein was speculated. The results of phenotypic analysis of solid plate show that the deletion of CaPHM7 gene affects the growth of Candida albicans with CaURA3 gene on solid plate. The results suggested that the gene might have genetic interaction with CaURA3 gene. The heterozygote deletion of CaPHM7 gene was sensitive to ketoconazole, while the homozygous deletion strain was more sensitive to ketoconazole than that of homozygous deletion strain. The results showed that the sensitivity of Candida albicans to ketoconazole was related to the dose of CaPHM7 gene, and the absence of CaPHM7 gene also led to the susceptibility of Candida albicans to surfactant SDS. The results of hyphal development analysis of the immunosuppressant rapamycin tolerance. 2) showed that the deletion of CaPHM7 gene resulted in the defect of mycelium development in the liquid YPD 10% fetal bovine serum Fetal Bovine serumn FBSS-Spider and Lee's medium, and the results showed that the absence of the CaPHM7 gene resulted in the hyphae development of Candida albicans in 10% fetal bovine serum. In addition, the deletion of CaPHM7 gene also resulted in the colony growth of Candida albicans on solid Spider and YPD 10S plates. The results of fluorescence localization analysis of mycelium development on solid Spider and Lee's plates showed that CaPhm7 existed on the plasma membrane of Candida albicans and hyphae cells. The results of functional complementation analysis of homologous protein of SDS, rapamycin and ketoconazole showed that CaPHM7 could not complement the homologous gene ScPHM7 in Saccharomyces cerevisiae cells. The function of zinc chloride and chlortetracycline is another homologous protein of AtCsc1 in Candida albicans. It has been found that CaSPO75 gene may negatively regulate cell wall pressure and terbinafine pressure. Positive regulation of ketoconazole pressure and its role in the homeostasis of zinc and manganese ions in Candida albicans.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R379.4

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