基于组学技术的黄曲霉毒素合成调控基因的筛选及功能验证

Aflatoxins are highly toxic and carcinogenic secondary metabolites produced mainly by Aspergillus flavus and Aspergillus parasiticus, recognized as the Class I carcinogens by World Health Organization(WHO). By now, the biosynthesis pathway of aflatoxin is relatively clear, however, as the regulation mechanisms of aflatoxin biosynthesis are not clear, the contamination problem is far from being solved. Although some regulatory genes such as aflR and aflS were discovered to play important roles in aflatoxin biosynthesis, some reports found expression levels of the discovered regulatory genes did not change between strains with different aflatoxin productions, indicating there are some new regulatory genes. Based on the 445 Aspergillus flavus strains with different aflatoxin productions isolated and identified in our lab, this project is aimed to use RNA-Seq, two-dimensional electrophoresis as well as the iTRAQ labeling technique to analyze the differences at RNA and protein levels among Aspergillus flavus strains with no, low and high aflatoxin productions under the same culture condition, select the candidate regulatory genes; verify their functions by the knocking out and overexpression techniques, thus to determine the key regulators, which will provide the theoretical foundation for aflatoxin control.

黄曲霉毒素是一类主要由黄曲霉和寄生曲霉真菌产生的次级代谢产物，被世界卫生组织认定为I级致癌物，严重影响人类健康。有效防控黄曲霉毒素的关键是解析毒素合成及其调控机理。目前，黄曲霉毒素的合成路径已比较清楚，但其调控机理还有待研究，虽找到了一些调控基因如aflR和aflS等，但有研究表明即使这些调控基因表达水平没有变化，菌株的产毒量仍有很大差异，提示还存在其它的调控基因。本项目将在申请者已分离鉴定到445株产毒量不同的黄曲霉基础上，采用RNA-Seq、二维电泳、iTRAQ标记的定量蛋白质组学技术分别从转录水平和蛋白质水平系统分析不产毒、低产毒和高产毒黄曲霉其RNA和蛋白质的表达变化情况，筛选出候选调控基因；采用基因敲除和过量表达方法研究基因对菌株产毒的影响，确定关键的调控基因，为黄曲霉毒素的防控提供理论依据。

黄曲霉毒素是一类主要由黄曲霉和寄生曲霉真菌产生的次级代谢产物，被世界卫生组织认定为I级致癌物，严重影响人类健康。有效防控黄曲霉毒素的关键是解析毒素合成及其调控机理。目前，黄曲霉毒素的合成路径已经比较清楚，但其调控机理还有待研究，虽找到了一些调控基因如aflR和aflS等，但有研究表明即使这些调控基因表达水平并没有变化，菌株的产毒两仍有很大差异，提示还存在其他的调控基因。本项目在申请者已分离鉴定到445株产毒量不同的黄曲霉基础上，采用RNA-seq组学技术从转录水平系统分析了来自同一地区（遗传背景较一致）的高产毒菌TF-12、中产毒菌TF-7和不产毒菌XinZ-16黄曲霉的基因表达变化情况，共鉴定到2554个差异表达基因，通过GO富集分析发现这些差异表达基因主要参与代谢、氧化还原、细胞防御等过程，其表达水平与黄曲霉毒素的合成调控密切相关。其中与氧化胁迫相关的调控黄曲霉毒素合成的Yap1同源基因AFLA_129340（AfapA）的表达差异极为显著。通过基因工程手段将AfapA基因敲除后，菌丝生长没有受到明显抑制，但是敲除菌株不能合成黄曲霉毒素。同时用不同浓度的过氧化氢处理黄曲霉菌株，发现不同氧化压力下黄曲霉毒素合成基因表达量有显著差异。该研究初步验证了一个新的够调控黄曲霉毒素合成的调控基因AfapA的功能，为黄曲霉毒素的防控研究提供线索。