丝裂原活化蛋白激酶SakA/AtfA通路介导活性氧对黄曲霉毒素B1合成的调控作用及分子机制研究

Aflatoxin B1 is a strong carcinogen, which can cause serious food safety problems. To clarify the regulation mechanism of aflatoxin B1 synthesis is the key to control mycotoxins contamination. The previous study found that mitogen-activated protein kinase SakA and transcription factor AtfA could influence AFB1 synthesis, but the regulatory effect and mechanism remains unclear. In this study, firstly, the function of SakA and AtfA in AFB1 synthesis is studied and the regulation effect of mitogen-activated protein kinase SakA/AtfA pathway mediated by reactive oxygen on AFB1 synthesis is elucidated. Secondly, to clarify the activation mechanism of SakA/AtfA pathway by reactive oxygen species, the phosphorylation sites of SakA and AtfA are predicted and validated; the activation effect of reactive oxygen on SakA and AtfA is studied from gene level, protein expression level, protein phosphorylation level and subcellular localization; the interaction between SakA and AtfA is also explored. Finally, to illuminate the effect of AtfA on regulatory effect of aflatoxin B1 synthesis, the aflatoxin B1 synthesis genes responsed to AtfA are predicted and validated; the effects of AtfA on transcription level of aflatoxin B1 synthesis genes and its role model were studied. Then, the regulatory mechanism of mitogen-activated protein kinase SakA/AtfA pathway mediated by reactive oxygen on aflatoxin B1 synthesis was revealed. This research can provide new targets for control of AFB1 contamination in food.

黄曲霉毒素B1（AFB1）是一种强致癌物，易引发严重食品安全问题，明晰其合成的调控机制是控制毒素污染的关键。前期研究发现丝裂原活化蛋白激酶SakA及其下游转录因子AtfA参与了活性氧对AFB1合成的激活，但作用机制不明。本项目拟①研究SakA与AtfA对AFB1合成的影响，解析SakA/AtfA通路在活性氧诱导AFB1合成中的功能；②预测并验证SakA和AtfA磷酸化位点，从基因转录、蛋白表达及磷酸化水平、亚细胞定位等角度考察活性氧对SakA和AtfA的作用，探究SakA与AtfA互作关系，阐明活性氧对SakA/AtfA通路的激活效应；③鉴定AFB1合成途经中响应AtfA调控的目的基因，研究AtfA对目的基因转录的影响及作用模式，阐明AtfA对AFB1合成的调控作用，揭示SakA/AtfA通路介导活性氧调控AFB1合成的分子机制，为控制食品中AFB1提供新靶标

黄曲霉毒素B1（AFB1）是由黄曲霉等真菌产生的一种强致癌性真菌毒素，ROS介导的氧化应激是其合成的先决条件。为明晰AFB1合成的调控机制，本项目首先通过RNA-seq技术分析不同ROS水平下黄曲霉转录组差异，明确了氧化胁迫诱导的SkaA/AtfA通路调控了AFB1的合成。基于黄曲霉转录组的数据分析，进一步研究了不同ROS水平下，细胞质与细胞核中激酶SakA蛋白表达及磷酸化水平、转录因子基因atfA及AFB1合成基因簇中aflA、aflC、aflE、aflJ、aflR的转录水平、AFB1及其前体物质NOR合成的差异。发现黄曲霉胞内ROS升高，将激活SakA，使其磷酸化，进入细胞核内激活转录因子AtfA活性，AtfA则进一步激活AFB1合成基因簇上aflA、aflC、aflE、aflJ、aflR基因的转录，诱导AFB1合成。为解析氧化胁迫下黄曲霉转录因子AtfA的功能，本项目利用同源重组技术构建AtfA的缺失突变株，分析敲除体在氧化胁迫下，其生长发育、产孢能力、孢子萌发、菌核形成、AFB1合成、侵染能力等表型的变化，发现敲除体对H2O2引起的氧化胁迫较为敏感，菌丝体生长明显受到抑制；atfA缺失导致黄曲霉孢子体积变小，萌发显著受到抑制，并阻止了黄曲霉菌核的生成，降低了AFB1的生物合成；同时，atfA缺失降低了黄曲霉孢子在花生、玉米中的侵染能力。最后利用Chip-seq技术发现AtfA调控的靶基因主要参与了孢子及菌丝生长调节、RNA聚合酶Ⅱ转录、细胞生长发育、MAPK级联反应、响应胞外压力胁迫、细胞代谢、细胞交流等功能，涉及到的通路有MAPK、脂肪酸合成、碳代谢、氨基酸合成、次级代谢物合成、GnRH、TCA循环等通路。本项目成果将为食品中AFB1的控制提供新靶标，保障食品安全。