白念珠菌14-3-3蛋白Bmh1在细胞生长和菌丝发育中的功能解析

Functions of 14-3-3 protein Bmh1 in cell growth and hyphal development of Candida albicans

摘要:

[目的]应用Tet-off启动子研究白念珠菌唯一的14-3-3蛋白Bmh1在白念珠菌生长和菌丝发育过程中的功能。[方法]在白念珠菌URA3⁺菌株SN152中，我们敲除了1个BMH1基因拷贝，并用Tet-off启动子替代另一个BMH1基因拷贝的启动子，得到了可以用强力霉素（Doxycycline）控制Bmh1表达水平的菌株。然后我们通过斑点试验和形态学观察对该菌株的生长和菌丝发育表型进行了分析。通过在ras1、flo8、efg1、cph1、tec1等重要菌丝发育调控因子突变体中过表达Bmh1，我们初步研究了Bmh1在菌丝发育调控网络中的位置。最后，我们构建了一些不同C末端的Bmh1嵌合体并检测了其对白念珠菌生长和菌丝发育的影响。[结果]Doxycycline诱导Bmh1表达水平下调时严重抑制了细胞的生长。非Doxycycline诱导条件下Bmh1高表达强烈促进了细胞的菌丝发育。这一促进作用绕过了ras1、efg1、cph1和tec1等基因缺失的影响，却被flo8基因的缺失阻断。C末端缺失或更换异源C末端的所有Bmh1突变株在Doxycycline诱导时都能够正常生长，但是没有明显促进菌丝发育。[结论]验证了白念珠菌14-3-3蛋白Bmh1是细胞生长所必需的，证明了Tet-off启动子可以严密控制Bmh1的表达水平。Bmh1是一个菌丝发育的正调控因子，位于Ras1、Efg1、Cph1和Tec1的下游，Flo8的上游。Bmh1的保守结构域是细胞生长所必需的，而C末端则是生长非必需的。

关键词:白念珠菌;14-3-3;Bmh1;菌丝发育

Abstract:

[Objective] We studied the roles of 14-3-3 protein Bmh1 of Candida albicans in its cell growth and hyphal development by using a Tet-off promoter. [Methods] Based on the URA3⁺ strain SN152 of C. albicans, we deleted one BMH1 allele and replaced the promoter of the other allele with the Tet-off promoter, generating a strain in which Bmh1 expression was controlled by Doxycycline. Then we explored the phenotypes of cell growth and hyphal development via spot analysis and morphological observations. Further, we preliminarily studied the position of Bmh1 in the network of hyphal development regulation via overexpression of Bmh1 in the mutants of ras1, flo8, efg1, cph1 and tec1, which were very important hyphal development regulators. Finally, we constructed some Bmh1 mutants with different C-terminals and detected their effects on cell growth and hyphal development of Candida albicans. [Results] Doxycycline-induced knockdown of Bmh1 inhibited cell growth severely. Highly-expressed Bmh1 improved hyphal development significantly without Doxycycline induction. This improvement bypassed the effects of ras1, efg1, cph1 and tec1 gene deletions and was blocked by flo8 deficiency. All Bmh1 mutants with C-terminal deletion or heterologous C-terminals grew normally with Doxycycline induction and the hyphal development was not improved obviously. [Conclusion] We validated that the 14-3-3 protein Bmh1 of Candida albicans is essential for cell growth, proving the tight controlling of the Tet-off promoter on Bmh1 expression. Bmh1 functions as a positive regulator of hyphal development and plays roles in the downstream of Ras1, Efg1, Cph1 and Tec1, and in the upstream of Flo8. The conserved domain of Bmh1 is required for cell growth while the C-terminal is not.

Key words:Candida albicans;14-3-3;Bmh1;hyphal development