Fus3-MAPK信号通路调控黄曲霉毒素合成的分子机制

黄曲霉毒素（AFT）具有强致癌性和强毒性，AFT污染严重威胁我国食品安全和出口贸易，造成巨大经济损失，控制AFT污染已成为保障食品安全、促进经济发展的国家重大需求。AFT合成调控机制是国际研究热点，是研发精准控制技术的关键前提；Fus3-MAPK信号通路是一类普遍存在于真菌中的丝氨酸/苏氨酸蛋白激酶信号通路，协调真菌有性发育和次级代谢，前期研究发现Fus3-MAPK通路相关基因敲除抑制黄曲霉中AFT合成，但具体调控机制仍不清楚。本项目拟：在前期研究基础上构建Fus3-MAPK信号通路中各激酶基因的敲除和回补菌株，研究各激酶基因对AFT合成的影响，采用转录组测序和qPCR等技术揭示各激酶基因对AFT合成的转录调控作用；特异性磷酸抗体和体外激酶分析实验检测敲除和回补株中各激酶的磷酸化水平，结合酵母启动子替代和双分子荧光互补解析各激酶功能，揭示各激酶磷酸化级联反应过程；荧光蛋白标记研究各激酶的亚细胞定位及共定位情况，双分子荧光互补检测胞内互作和复合物形成，延时图像或视频解析激酶的移动规律及核转运过程；串联亲和纯化鉴定Fus3互作蛋白，双分子荧光互补、激酶分析和磷酸抗体检测验证蛋白互作和磷酸化过程，确定Fus3特异性磷酸化的靶标蛋白，解析靶标蛋白对AFT合成的调控作用。完成上述研究将明确Fus3-MAPK信号通路各激酶的蛋白互作关系和磷酸化级联反应过程，确定各激酶的亚细胞定位及共定位情况，揭示各激酶在细胞内的移动规律及核转运过程，确定Fus3特异性磷酸化的靶标蛋白并明确其磷酸传递过程，揭示靶标蛋白对AFT合成的调控作用，最终阐明Fus3-MAPK信号通路调控AFT合成的分子机制。项目研究结果将进一步完善AFT合成调控网络，为阻断AFT合成、研发精准控制技术提供靶点和线索，为减轻AFT污染、保障食品安全提供理论依据和技术支撑，既具有重大科学意义，又有明确应用前景。

Aflatoxins (AFT) are highly carcinogenic and toxic and seriously threaten food safety and export trade in China. AFT synthesis and its regulatory mechanism are hot topics worldwide and a key prerequisite for the development of precision control techniques. The Fus3-MAPK signaling pathway is a serine/threonine protein kinase signaling pathway commonly found in fungi that coordinates both fungal sexual development and secondary metabolism. Previous studies have found that the knockout of genes in the Fus3-MAPK pathway resulted in the inhibition of AFT biosynthesis. However, the molecular mechanism of regulation remains unclear. Firstly, based on previous studies, knockout and complementation strains of kinase gene in Fus3-MAPK signaling pathway are constructed. The effects of these genes on the transcription levels of genes related with AFT biosynthesis are investigated, and the transcriptional regulation of each kinase gene on AFT biosynthesis is clarified by RNA-seq and qPCR techniques. Secondly, the phosphorylation level of each kinase in the deletion and complementation strains is determined by specific phospho-antibody and in vitro kinase assays. The functions of these kinases are analyzed using yeast promoter substitution and bimolecular fluorescence complementation (BiFC) assay, revealing the phosphorylation process of these kinases. Thirdly, the subcellular localization and co-localization of these kinases are investigated using fluorescence protein. Intracellular interaction and complex formation are detected by BiFC assay, and migration and nuclear transport are determined using time-lapse images and time-lapse movies. Finally, the interaction partners of Fus3 are purified and identified by tandem affinity purification. Then, BiFC assay, kinase analysis, and phosphoantibody assays validate protein interactions and phosphorylation processes. Thus, the specific target proteins of Fus3 are identified, and the regulation of the target proteins on AFT biosynthesis is clarified. This study can elucidate the protein interaction and phosphorylation cascade of each kinase in the Fus3-MAPK signaling pathway, and the subcellular localization and colocalization of each kinase, revealing the movement pattern and nuclear transport process of each kinase. The target protein for Fus3-specific phosphorylation can be identified, its phosphorylation process is verified, and the regulation of AFT synthesis by the target protein is revealed, and eventually elucidating the molecular regulatory mechanism of AFT biosynthesis by Fus3-MAPK pathway. This will provide theoretical basis for improving the regulation network of AFT biosynthesis and developing new targets for the prevention and control of AFT.