【摘要】 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。更多还原

【Abstract】 In Candida albicans,adaptation to environmental pH is relevant to its pathogenicity.Calcium signaling pathway involves in many stress responses and often accompany with Ca2+ fluctuation.We constructed CCH1 and MID1 mutant strains and studied their effect on calcium influx and further investigated the regulation by Crz1p transcription factor.We used PCR-directed gene disruption to construct cch1Δ/Δ and mid1Δ/Δ null mutant.By using a flow cytometry-based method we monitored the free cytosolic Ca2+ levels under alkaline stress.Moreover,we constructed pPHO89-LacZ plasmids and by β-Galactosidase assays,we analyzed the changes of LacZ activities after gene disruption.The results showed that alkaline stress induced calcium burst reduced obviously in cch1Δ/Δ and mid1Δ/Δ mutant strains,also for LacZ activities,and fully abolished in crz1Δ/Δ mutant strain.Finally,by realtime PCR,we confirmed the regulation role of Crz1p in CCH1 and MID1 genes but in a calcineurin independent way.Studies on the effect of calcium pathway on response to alkaline stress will provide an important theoretical basis for Candida albicans infection-oriented treatment and new drug targets.更多还原