几种白念珠菌高铁还原酶基因的功能研究

Characterization of Candida albicans ferric reductase genes in response to environmental stresses

摘要:

摘要:【目的】高铁还原酶在白念珠菌铁离子获得过程中发挥着重要作用。通过研究高铁还原酶基因的功能和表达调控，揭示其不同的环境应答策略。【方法】通过Northern 杂交的方法分析不同低铁环境对高铁还原酶基因表达水平的影响。构建高铁还原酶基因缺失菌株，探究基因缺失后对细胞表面高铁还原酶活力和生长状况的影响。利用激光共聚焦显微镜观察Frp1蛋白的细胞学定位。【结果】酸性条件显著上调FRE10基因的表达水平，而碱性条件能显著提高FRE2基因的表达量。在酸性条件下，FRE10基因的缺失会显著地下调细胞表面高铁还原酶活力。在碱性条件下，fre2Δ/Δ缺失菌株表现出严重缺陷的生长能力和显著降低的表面高铁还原酶活力。细胞学定位实验发现Frp1蛋白位于液泡中。【结论】FRE2和FRE10基因的表达模式主要是酸碱依赖性的。Fre2是碱性条件下高铁还原酶活力的主要贡献者。Frp1蛋白位于液泡中，在液泡内储存铁的活化和转运过程中可能发挥重要作用。

关键词:关键词:白色念珠菌，高铁还原酶基因，FRE2，FRP1

Abstract:

Abstract:［Objective］Ferric reductases play a central role in iron acquisition and mobilization in C．albicans．This study focuses on stress response strategies exhibited by several ferric reductase genes through function and expression analyses．［Methods］ Northern blot analysis was used to examine ferric reductase genes expression levels in different iron deficiency．We constructed ferric reductase-null mutants by a PCR-based homologous recombination，and examined the effects of gene deletion on cell-surface ferric reductase activity and growth ability under different conditions． Sub-cellular localization of Frp1-GFP fusion was imaged and analyzed by confocal laser scanning fluorescence microscopy． ［Ｒesults］FRE10 was highly expressed at acidic pH，compared to that at alkaline pH，whereas the expression of FＲE2 was just the opposite． Deletion of FＲE10 resulted in a significant decreased surface reductase activity at acidic pH，with 75. 5% downregulation compared to wild-type levels．The fre2Δ/Δ mutant showed significantly attenuated growth ability and cellsurface ferric reductase activity at alkaline pH．Sub-cellular localization revealed that the green fluorescence was accumulated in the vacuoles．［Conclusion］The expression of both FRE10 and FRE2 is induced in a pH-dependent manner．FRE2 encodes a major cell surface ferric reductase under alkaline pH condition．Frp1 localizes to the vacuole，and might support mobilization and transport of vacuolar ferric iron stores．

Key words:Keywords: Candida albicans，ferric reductase gene，FRE2，FRP1