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白念珠菌Candida albicans对环境pH的适应能力与其致病性有密切关系,钙信号转导途径介导许多环境压力的应答并伴随胞内钙离子浓度的瞬间变化。通过构建钙通道基因CCH1和MID1的缺失突变株,在碱性pH条件下,研究其对胞内钙内流的影响以及转录因子Crz1p对CCH1和MID1基因的调控作用。使用二步法PCR介导的基因敲除技术构建cch1?/?和mid1?/?突变菌株,利用流式细胞术比较野生型和突变型菌株在碱性pH条件刺激下胞内钙的瞬间变化,进一步构建pPHO89-LacZ重组质粒并利用β-半乳糖苷酶体系分析CCH1和MID1基因缺失对外钙内流以及转录因子Crz1p对LacZ活性的影响,结果发现碱性pH条件引起的钙离子浓度的瞬间变化在cch1?/?和mid1?/?缺失突变株中明显减弱,LacZ活性也显著降低,且在crz1?/?缺失突变株中完全消失。最后利用实时定量PCR,确定转录因子Crz1p在碱性pH条件下对CCH1和MID1具有非钙调神经磷酸酶依赖性的调控作用。对钙信号途径在碱性pH压力应答中作用的研究,将为临床上白念珠菌感染的治疗和新药靶点导向提供重要的理论依据。
The Candida albicans adaptation to environmental pH is closely related to its pathogenicity. The calcium signaling pathway mediates many environmental stress responses with transient changes in intracellular calcium concentration. The effects of Crz1p on the regulation of CCH1 and MID1 genes were studied by constructing calcium channel CCH1 and MID1 deletion mutants under alkaline pH conditions. Two-step PCR-mediated gene knockout technique was used to construct cch1? /? And mid1? /? Mutant strains. Flow cytometry was used to compare transient changes of intracellular calcium in wild-type and mutant strains stimulated with alkaline pH The recombinant plasmids pPHO89-LacZ were further constructed and the effects of CCH1 and MID1 gene deletion on the calcium influx and the transcription factor Crz1p on the LacZ activity were analyzed by β-galactosidase system. The results showed that the alkaline pH condition caused the instantaneous calcium concentration Changes in cch1? /? And mid1? /? Deletion mutant was significantly weakened, LacZ activity was also significantly reduced, and in crz1? /? Deletion mutant completely disappeared. Finally, real-time quantitative PCR was used to determine that the transcription factor Crz1p has a non-calcineurin-dependent regulation of CCH1 and MID1 at alkaline pH. The study of the role of calcium signaling pathway in alkaline pH pressure response will provide important theoretical basis for clinical treatment of Candida albicans infection and new drug target orientation.