肉桂醛抑制黄曲霉毒素生物合成的分子调控机理

Aflatoxins (AFT) are carcinogenic, genotoxic and immunotoxic. Foods and feeds are generally contaminated with AFT, which become a great health threaten to humans and animals. In our previous study, we found that at lower concentration, the reduction of AFT production by cinnamaldehyde was due to down-regulation of AFT biosynthesis. But the key regulatory genes and their regulatory pathways of action were unclear. In the present study, RNA-seq, two-dimensional electrophoresis as well as iTRAQ labeling techniques will be used to analyze the difference at RNA and protein levels in Aspergillus flavus with cinnamaldehyde or without. Then the candidate regulatory genes will be selected. Then the mutants of the regulatory genes knocked out will be constructed. The effect of regulator genes on the expression of AFT biosynthetic regulatory genes (aflR and aflS) and structural genes and AFT production will be analyzed. The key regulatory genes and their functions will be determined. The interactions of regulatory genes and AFT biosynthetic genes will be identified by yeast two-hybrid assay, and the regulatory mechanism of the regulatory genes on AFT biosynthetic genes will be elucidated. The gene regulatory pathway of inhibition of AFT biosynthesis by cinnamaldehyde will be constructed. This research can preliminarily reveal the regulatory mechanism of inhibition of AFT biosynthesis by cinnamaldehyde. And this research is also significant for usage of cinnamaldehyde to control AFT contamination in foods and feeds.

黄曲霉毒素（AFT）是普遍污染食品和饲料的强毒性和致癌性物质，严重威胁人畜健康。本项目前期研究发现，低浓度肉桂醛可以调控AFT生物合成而抑制毒素产生，但关键调控基因及其调控路径不清楚。在此基础上，本项目拟采用RNA-seq、二维电泳和iTRAQ定量蛋白质组学等技术，分析肉桂醛处理下黄曲霉RNA和蛋白的差异表达，筛选参与肉桂醛抑制AFT生物合成的候选调控基因；构建调控基因敲除突变体，分析候选调控基因对AFT生物合成基因簇内关键调控基因（aflR和aflS）和合成基因表达以及AFT产量的影响，确定关键调控基因及其功能；通过酵母双杂交技术鉴定调控基因与AFT生物合成关键基因的相互作用，揭示调控基因对AFT合成基因的调控机制，明确肉桂醛抑制AFT生物合成的基因调控路径。上述研究将初步阐明肉桂醛阻断和抑制AFT生物合成的分子调控机理，为利用肉桂醛防控AFT污染食品和饲料提供理论基础。

黄曲霉毒素（AFs）是普遍污染食品和饲料的强毒性和强致癌性物质，严重威胁食品安全和人畜健康，AFs污染绿色防控剂研发及其调控机制研究一直是国际食品安全研究的热点。前期研究发现，低浓度肉桂醛可以调控AFs合成而抑制毒素产生，但关键调控基因及调控路径不清楚。为此，本项目采用RNA-seq和RT-qPCR等技术，明确了0.6mM肉桂醛对黄曲霉中AFs合成、氧化应激、脂肪酸β-氧化、生长发育、孢子形成等相关基因转录的调控作用；发现肉桂醛调控氧化应激转录因子表达，激活抗氧化系统，降低胞内ROS水平，并继而下调毒素合成基因簇的表达，最终抑制AFs的生物合成。明确了参与肉桂醛抑制AFs合成的关键调控基因：Velvet复合体相关基因fluG、应激转录因子srrA、bZIP转录因子ap-1、C2H2锌指类转录因子msnA、CREB/ATF成员atfB、Fus3-MAPK信号通路基因等。通过基因敲除回补，揭示了转录因子ap-1、Fus3-MAPK信号通路各激酶基因对AFs合成及其基因转录的正调控作用；采用酵母双杂交实验，重点研究揭示了Fus3-MAPK信号通路各激酶之间的蛋白互作；综合分析研究结果，勾勒出了肉桂醛抑制AFs生物合成的转录调控途径，阐明了肉桂醛抑制AFs生物合成的分子调控机理。在机制解析的基础上，本项目利用肉桂醛、柠檬醛和丁香酚等复配了复合植物精油防霉剂，开展了为期一年的玉米储藏防霉实验，结果表明该防霉剂可高效抑制黄曲霉等真菌生长和毒素产生，并能保持玉米品质，防止品质劣变。项目研究结果为今后进一步深入解析AFs合成调控机制和发掘防控新靶点提供理论基础；此外，本项目明确了肉桂醛等精油类物质能够有效降低仓储粮食真菌毒素污染，为植物精油类物质在毒素防控领域的应用提供了科学依据。项目已发表SCI论文12篇、中文核心1篇，申请发明专利4项，作为部分成果获2016年中国农科院杰出科技创新奖。