调控黄曲霉毒素合成及氧化应激双功能转录因子的鉴定与功能研究

The regulation of aflatoxin biosynthesis has become a model in elucidating the regulation of secondary metabolite biosynthesis in micro-organisms. It had been showed that there was a strong relationship between the biosynthesis of secondary metabolism and the intracellular oxidative stress in fungi, and the transcription factors that response to oxidative stress played an important role in regulating the secondary metabolite biosynthesis. The secondary metabolism was also considered as the secondary line of the defense to oxidative stress. Our previous studies showed that the aflatoxin biosynthesis was dramatically inhibited and the activities of the oxidative stress related enzymes were greatly changed after Aspergillus flavus was treated with the antioxidant gallic acid. We also found that 20 genes, encoding transcription factor with unknown function were significantly differently transcribed after treatment with gallic acid, which indicated that these transcription factors may play an important role in regulating aflatoxin biosynthesis. This project will depart from these 20 genes for identifying transcription factors with double-function in regulating the aflatoxin biosynthesis and response to oxidative stress. We will focus on exploring the functions of 2 to 3 novel double-functional transcription factors and their target genes by using the technology of molecular biology and genetics. Finally, we will construct the regulatory network coordinating the expression of genes involved in aflatoxin biosynthesis and the oxidative stress in A. flavus. This study will provide a theoretical basis for elucidating the regulatory mechanism of the aflatoxin biosynthesis and its relationship with oxidative stress.

黄曲霉毒素生物合成调控机制的研究已成为微生物次级代谢调控研究的模型。研究表明，真菌次级代谢与氧化压力的细胞响应之间存在密切关联，应对氧化压力的转录因子也参与了对次级代谢的调控。我们的前期研究表明，抗氧化剂没食子酸处理黄曲霉后，黄曲霉毒素的合成受到抑制及引起氧化应激相关酶活性的改变，并且造成20个未知功能转录因子基因的转录发生了显著差异表达。本项目从这20个转录因子基因出发，以黄曲霉毒素生物合成调控网络为中心，通过系列分子生物学和分子遗传学技术如基因敲除、转录组测序、靶基因筛选等，鉴定出在黄曲霉毒素生物合成及应对氧化压力中起双重调控功能的转录因子，重点研究2-3个关键双功能转录因子及其靶标基因在黄曲霉毒素合成代谢及应对氧化压力中的功能，并在此基础上阐明黄曲霉在应对氧化压力时AF合成的胞内信号转导途径。本项目将为最终揭示黄曲霉毒素生物合成调控机制及其与氧化应激的共调控机制提供理论基础。

黄曲霉毒素为具极强致癌性的真菌次级代谢物，对其生物合成代谢调控机制的研究已成为微生物次级代谢调控研究的热点，也是解决黄曲霉毒素污染的根本途径。本研究在进一步利用转录组技术对不同浓度抗氧化剂没食子酸处理黄曲霉后的转录组数据进行了深入挖掘，在此基础上，重点对黄曲霉毒素合成、黄曲霉发育及氧化应激相关的转录因子FarB、CreA、Ham系列、SfgA以及对毒素合成和黄曲霉发育有调控作用的DNA甲基转移酶同源基因dmtA在黄曲霉次级代谢和发育（包括孢子、菌核、菌丝）中的作用机制开展了深入研究。发现：（1）转录因子FarB和CreA调控黄曲霉毒素合成和氧化应激，没食子酸处理降低了黄曲霉胞内的氧化压力水平，并通过转录因子FarB调控黄曲霉胞内的脂肪酸β氧化，进而影响黄曲霉毒素的合成，同时，黄曲霉毒素合成代谢基因簇基因在0.8%没食子酸处理后显著下调表达与碳代谢物阻遏相关基因creA的下调表达相关，creA基因的敲除菌株显著抑制了黄曲霉毒素的产生，黄曲霉中CreA通过影响黄曲霉毒素合成代谢基因簇基因的表达实现对黄曲霉毒素合成的调控，该部分结果发表在《Toxins》上（2018）。（2）Ham系列调控因子（HamE-I）与LaeA互作调控黄曲霉毒素合成和菌核发育，且5个ham基因（hamE-I）的表达均受到LaeA的正向调控，该部分结果发表在《Frontiers in Microbiology》上（2017）。（3）调控因子SfgA对黄曲霉次级代谢和生长发育具有多向调控功能，如SfgA对黄曲霉毒素和胶霉毒素合成起负调控作用而对曲酸的合成则起正调控作用，该部分结果正在投稿中。（4）发现DNA甲基转移酶唯一的同源基因dmtA对黄曲霉毒素合成及其环境适应性具重要作用，dmtA基因的敲除影响黄曲霉毒素合成、黄曲霉孢子发育并导致抗逆结构菌核的消失，同时降低了黄曲霉对渗透压、低氧、低水活及高钙离子环境压力的敏感性，该部分结果发表在《Fungal Biology》上（2017）。（5）对所研究相关基因在黄曲霉毒素生物合成及胞内氧化应激调控中的作用通路进行了阐述。总之，该研究为黄曲霉毒素合成代谢调控机制的揭示提供了一定的理论基础。