二甲基三硫抑制黄曲霉菌生长和产毒的分子机制研究

Aspergillus flavus and the produced highly carcinogenic aflatoxins cause great damage to crop production and human health. Nowadays the control of this pathogen and aflatoxins at pre- and post-harvest has been being a hot research topic. Acetyl-CoA carboxylase (ACC) catalyzed the carboxylation of acetyl-CoA to produce malonyl-CoA, which is the first and speed-determining step in fatty acid biosynthesis pathway (for cell membrane biosynthesis) and aflatoxins biosynthesis pathway. ACC plays an important role in the growth and aflatoxins production of A. flavus. It has been proved that one marine bacterial strain YM8 was effective in inhibiting the growth of A. flavus and aflatoxins production by the emitting of volatile dimethyl trisulfide (DMTS). DMTS can dramatically inhibit the enzyme activity of ACC, and significantly reduce the expression of ACC and other important genes in fatty acid biosynthesis pathway and aflatoxins biosynthesis pathway, and further prevent the growth and aflatoxins production. Hence, the enzyme activity of ACC is the potential inhibitory mechanism of DMTS. Based on that we will construct the mutant of ACC enzyme, and reveal the same changes at the characters of phenotype, cell ultra-structure, gene expression, enzyme activity and metabolite production activity with the A. flavus treated with DMTS. Finally, we will reveal that ACC enzyme is the main inhibitory mechanism of DMTS against A. flavus and aflatoxin. The completion of this project will be helpful for the exploitation of novel biocontrol agents.

黄曲霉菌及其产生的强致癌性黄曲霉毒素对粮食生产和人类健康危害严重，其田间和储藏期的安全防控是目前研究的热点。乙酰辅酶A羧化酶（ACC酶）催化乙酰辅酶A转化为丙二酰辅酶A，是脂肪酸生物合成（细胞膜合成）和黄曲霉毒素生物合成通路的首步反应和限速步骤，在黄曲霉菌的生长和产毒中至关重要。海洋细菌YM8产二甲基三硫（DMTS），可高效抑制储藏期黄曲霉菌的生长和黄曲霉毒素的产生，酶活性与转录组学分析表明，DMTS显著抑制了ACC酶的活性，及其参与的脂肪酸生物合成和黄曲霉毒素生物合成中关键基因的表达，ACC酶活性受抑制是黄曲霉菌停止生长和产毒的关键原因，为DMTS抑菌的潜在分子机制。本项目通过构建黄曲霉菌ACC酶功能缺失突变体，验证其与DMTS处理黄曲霉菌，在表型、细胞结构、基因表达、酶活性和代谢水平的相同变化规律，揭示ACC酶为DMTS抑菌的关键分子靶点，本项目的完成为新型生防菌剂的研发提供重要依据。

黄曲霉菌可侵染粮食和油料作物，并产生强致癌性黄曲霉毒素，对食品安全和人类健康危害严重，其安全防控，是目前急需解决的关键问题。研究结果显示，乙酰辅酶A羧化酶（Acetyl-CoA carboxylase, ACC）催化乙酰辅酶A转化为丙二酰辅酶A，是脂肪酸生物合成和黄曲霉毒素生物合成的首步反应和限速步骤，对黄曲霉菌的生长（细胞膜合成）和毒素的产生至关重要。海洋菌 YM8 产生的小分子物质二甲基三硫（DMTS），能够结合ACC酶，使酶活性由377.62降至191.42U/g ，显著下调脂肪酸生物合成和黄曲霉毒素生物合成途径中的关键基因表达量，并最终抑制菌丝生长和毒素的合成。由此推断，ACC酶可能为DMTS抑制黄曲霉菌生长和产毒的作用靶点。实验证明ACC基因为致死基因，在黄曲霉菌和禾谷镰刀菌中无法获得基因缺失突变体，进而采用同源建模和分子对接实验，证明DMTS与ACC酶存在互做，有5个结合位点，41个潜在的互做氨基酸靶点，且多数氨基酸位点在多种不同真菌生物中具有高度保守性，与DMTS的广谱抑菌性相验证。互做氨基酸位点的获得，为鉴定DMTS抑菌分子靶点的提供数据支持.此外，课题组5株新型的抑制储藏期黄曲霉菌生长和产毒的功能微生物，并鉴定功能活性物质，验证抑菌作用效果，相关成果已发表论文并申请国家发明专利，为新型生物抑菌剂的开发和应用提供新的材料。

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