高渗胁迫下应激活性蛋白激酶afSakA对黄曲霉毒素合成的调控机制研究

Aspergillus flavus is a pathogenic fungi distributed worldwide. It produces aflatoxin that can lead to aspergillosis and liver cancer, seriously threatening the health of human and animals. Researches have indicated that the biosynthesis of aflatoxin was affected by many factors. Among them, water activity is one of the most important environmental factors. However, the relationship between Hog pathway and the aflatoxin production under the high osmotic stress has not been reported. In this study, the results of preliminary test indicated that growth and aflatoxin biosynthesis of A. flavus was significantly influenced by the knockout of afsakA gene. In order to reveal the molecular mechanism of afSakA regulating aflatoxin synthesis under the osmotic stress further, subcellular location of MpkC and afSakA was assessed by GFP fusion expression. And the phosphorylation state of MpkA was determined by Western blotting. Then the mouse survival experiments were carried out to analysis the change of virulence. Therefore, this study will not only provide a new way for the study of the high osmotic response mechanism of A. flavus and the mechanism of aflatoxin synthesis, but also provide a method to control the pollution of A. flavus.

黄曲霉是世界性分布的致病性真菌，它产生的黄曲霉毒素可以导致曲霉病及肝癌等，严重危害人类和动物的健康。研究表明，黄曲霉毒素的产生受到多种因素影响，水分活度是最重要的环境因子之一。但是，高渗胁迫相关的Hog通路与黄曲霉产毒的关系的研究目前尚未见报道。本研究通过敲除黄曲霉应激活性激酶的基因afsakA，初步验证了该途径能显著影响黄曲霉的生长和产毒。为进一步揭示afsakA在高渗条件下调控黄曲霉毒素产生的分子机制，本研究拟采用GFP融合表达法对黄曲霉afsakA及相关mpkC基因进行亚细胞定位；用Western blotting分析MpkA磷酸化水平的变化；并采用小鼠毒性实验分析其对黄曲霉产毒的影响，阐明afsakA在高渗条件下如何与相关基因协调控制产毒过程。因此，本研究不仅将为深入研究黄曲霉的高渗响应机制和毒素产生机制作很好的铺垫，还可为黄曲霉污染的控制提供新的思路。

黄曲霉是世界性分布的致病性真菌，它产生的黄曲霉毒素可以导致曲霉病及肝癌等，严重危害人类和动物的健康。研究表明，黄曲霉毒素的产生受到多种因素影响，水分活度是最重要的环境因子之一。但是，高渗胁迫相关的Hog通路与黄曲霉产毒的关系的研究目前尚未见报道。本研究通过敲除黄曲霉应激活性激酶的基因afsakA，研究了高渗条件下黄曲霉Hog通路中afsakA在生理调控中的作用。研究发现，afsakA在高渗胁迫条件下，在黄曲霉产孢、生长、产毒、致病性等生理调控过程中都起到了重要作用。采用mcherry融合表达法对黄曲霉afSakA进行了亚细胞定位发现，在无胁迫情况下，afSakA在细胞质中表达；当处于渗透胁迫时进入细胞核内表达。用Western blotting分析菌株内激酶磷酸化水平的变化发现，突变体菌株在NaCl胁迫下体内均仍存在磷酸化激酶蛋白。本研究结果可为深入研究黄曲霉的高渗响应机制和毒素产生机制做很好的铺垫，还可为黄曲霉污染的防控提供新的思路。