白念珠菌CaRCH1基因的功能研究

发布时间：2018-05-11 10:47

本文选题：白念珠菌 + CaRCH1　；参考：《天津大学》2010年硕士论文

【摘要】：白念珠菌RCH1基因的ORF.长为1236 bp,编码一个411个氨基酸的蛋白,由于该基因的缺失株对氟康唑有抗性表型,所以曾经命名为CaFSP1。在课题研究中,我们发现rch1Δ/rch1Δ缺失株和pmc1Δ/pmc1Δ缺失株具有类似的表型,都对钙离子敏感,对LiCl和氟康唑有抗性的表型。我们知道环孢霉素A(CsA)可以抑制钙调磷酸酶,阻断白念珠菌Ca~(2+)/钙调磷酸酶途径。于是,首先我们将CsA加入CaCl_2的固体培养基中,rch1Δ/rch1Δ缺失株可以回复到野生型的生长状态。第二,胞外低浓度的Mg~(2+)能够抑制pmc1Δ/pmc1Δ缺失株对钙离子的敏感表型,我们将Mg~(2+)加入CaCl_2培养基中,rch1Δ/rch1Δ缺失株对CaCl2敏感的表型消失。第三,我们构建了pmc1Δ/pmc1Δrch1Δ/rch1Δ双基因缺失株,发现该缺失株与pmc1Δ/pmc1Δ缺失株相比,对钙离子更加敏感,而对LiCl和氟康唑的表型没有明显的差异。此外,钙调磷酸酶影响白念珠菌菌落的形态和菌丝的形成,我们发现pmc1Δ/pmc1Δrch1Δ/rch1Δ双基因缺失株在血清诱导下,菌丝的形成受到抑制。然而,在体内实验中,我们发现白念珠菌仅缺失PMC1基因,就会大大减弱细胞的毒力。最后,我们又构建了pCK-RCH1质粒,该质粒可以过量表达RCH1基因,将其转入pmc1Δ/pmc1Δ缺失株中,但未能弥补pmc1Δ/pmc1Δ缺失株的表型。以上实验可以说明CaRch1p具有调节细胞内钙离子稳态的作用,因此我们将该基因重新命名为CaRCH1即C. albicans regulator of calcium homeostasis。
[Abstract]:ORF of RCH1 gene of Candida albicans. The protein, which is 1236 BP long and encodes a 411-amino acid protein, was once named CaFSP1 because of its resistant phenotype to fluconazole. In our study, we found that rch1 螖 / rch1 螖 deletion strain and pmc1 螖 / pmc1 螖 deletion strain had similar phenotypes, which were sensitive to calcium ions and resistant to LiCl and fluconazole. It is known that cyclosporin A can inhibit calmodulase and block the Ca~(2 / Ca 2 + pathway of Candida albicans. So, first, we added CsA to CaCl_2 solid medium and the missing strain of rch1 螖 / rch1 螖 could return to the growth state of wild type. Secondly, the low concentration of Mg~(2 could inhibit the sensitive phenotype of pmc1 螖 / pmc1 螖 deletion strain to Ca ~ (2 +). Mg~(2) was added to CaCl_2 medium to eliminate the CaCl2 sensitive phenotype of rch1 螖 / rch1 螖 deletion strain. Thirdly, we constructed pmc1 螖 / pmc1 螖 rch1 螖 / rch1 螖 double gene deletion strain. It was found that the deletion strain was more sensitive to calcium than that of pmc1 螖 / pmc1 螖 deletion strain, but had no obvious difference in phenotype of LiCl and fluconazole. In addition, calmodulase affected the morphology and mycelium formation of Candida albicans colony. We found that the formation of hyphae was inhibited by pmc1 螖 / pmc1 螖 rch1 螖 / rch1 螖 double gene deletion strain induced by serum. However, in vivo experiments, we found that Candida albicans can significantly reduce the virulence of cells by simply missing the PMC1 gene. Finally, we constructed the pCK-RCH1 plasmid, which can overexpress the RCH1 gene and transfer it into the pmc1 螖 / pmc1 螖 deletion strain, but it can not make up for the phenotype of the pmc1 螖 / pmc1 螖 deletion strain. These results suggest that CaRch1p can regulate intracellular calcium homeostasis, so we rename the gene CaRCH1 or C. albicans regulator of calcium homeostasis..
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R379

【引证文献】