一氧化氮降解酶Fhb1调控黄曲霉毒素合成的分子机制研究

The ubiquitous fungus Aspergillus flavus, as a worldwide pathogen, produces the carcinogenic mycotoxin Aflatoxin (AF), which causes enormous agricultural economic losses and health problems at several levels worldwide. Our former study demonstrated that the disruption of Fhb1, an enzyme that is responsible for nitric oxide (NO) degradation, promoted the concentration of NO in A. flavus and inhibited AF biosynthesis. A multi protein modification sites including phosphorylation, acetylation and succinylation were also identified in this protein, suggesting that the synthesis and degradation of NO were regulated under a complex mechanism. In order to further investigate the molecular mechanism of Fhb1 on regulation of A. flavus development and AF biosynthesis, gene knock-out, over-expression and point mutation strategies will be performed to reveal Fhb1 functions in regulating AF biosynthesis. Gene cloning and proteins purification will be used to assay the protein activity in vitro as well. Then the LC-ESI-MS/MS is going to be performed for identification of the potential substrates for S-nitrosation induced by NO, which is related with AF biosynthesis. Gene knock-out and point mutation strategy will be further applied to reveal S-nitrosation functions in regulation of AF biosynthesis. Therefore, this study will not only firstly reveal the NO degrading enzyme Fhb1 is involved in the A. flavus development and AF producing, but could also promise to provide valuable information for new control strategies of AF contamination.

黄曲霉是一种世界性分布的致病菌，它产生的强致癌物黄曲霉毒素（AF），给农业生产造成巨大经济损失，其引发的食品安全问题也给人类健康带来巨大威胁。申请人前期研究发现黄曲霉中一氧化氮（NO）降解酶Fhb1的敲除抑制AF的生物合成；同时发现Fhb1蛋白上存在多种翻译后修饰位点，这些暗示了黄曲霉在合成和降解NO过程受到复杂的调控。为了深入研究Fhb1蛋白对黄曲霉发育和AF合成的调控作用及其潜在的分子机制，本研究拟以黄曲霉为材料，1）结合体外酶活实验和基因敲除、过表达、点突变等方法研究Fhb1的遗传学功能；2）通过LC-ESI-MS/MS手段鉴定黄曲霉菌中NO诱导的和AF合成相关的S-亚硝基化靶蛋白；3）结合基因敲除和点突变等方法深入研究这些靶蛋白上S-亚硝基化水平对AF合成的影响。预期结果将从新角度解释AF生物合成的调控机制，为黄曲霉及毒素的早期防控提供坚实的理论基础。

黄曲霉和其产生的黄曲霉毒素（AF）给农业生产造成巨大经济损失，并严重威胁人畜健康。前期研究发现黄曲霉中一氧化氮（NO）降解酶Fhb1的敲除抑制AF的生物合成；同时发现Fhb1蛋白上存在多种翻译后修饰位点。本研究通过基因敲除、点突变等基因操作手段发现一氧化氮降解酶Fhb1蛋白和组成型NO合成酶参与黄曲霉胞内NO浓度的调控，并且证实NO在黄曲霉毒素合成中发挥负调控作用。为研究NO对黄曲霉发育和毒素合成的调控机制，本研究结合Biotin switch方法和iodoTMT标记技术筛选和鉴定NO诱导的S-亚硝基化的蛋白，并首次在黄曲霉菌中成功鉴定到了394个亚硝基化蛋白，发现其参与细胞的多个过程。进一步筛选找到了参与TCA三羧酸循环通路中的多个亚硝基化靶蛋白。通过基因敲除等方法研究发现，NO可以诱导TCA循环中顺乌头酸酶等蛋白发生亚硝基化，从而抑制其酶活，导致TCA循环受阻，并使线粒体中NADH和ROS合成受阻，最终抑制黄曲霉毒素合成。本研究的发现揭示了黄曲霉毒素新的合成机制，并将为黄曲霉和黄曲霉毒素的防治提供重要的理论依据。

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