磷酸激酶slt2调控黄曲霉毒素产生的分子机制

Aspergillus flavus can widely pollute environment for human survival, and it produces aflatoxins that can lead to liver cancer, seriously threating the food safety and human health. Researches have shown that the aflatoxin generation was regulated by multiple signaling pathways, but regulation pathway mediated by the phosphokinase slt2 has not been reported. The preliminary research through the slt2 knockout confirmed that slt2 could significantly influence growth and aflatoxin biosynthesis of A. flavus. In order to further revealing the molecular mechanism of slt2 regulating aflatoxin biosynthesis, this study plans to knockout various functional domains of slt2, understanding specific roles of these domains. At the same time, transcription factors regulated by slt2 are knockout to determine the slt2 regulate way. Finally, this study will also conduct EMSA and yeast single hybridization technique to determine the specific sequence of toxigenic gene bound by transcription factors. Therefore, the study will not only first reveal the phosphokinase slt2 mediated signal pathway in the aflatoxin producing, but could also promise to provide new method for control the aflatoxin contamination.

黄曲霉能广泛污染人类生存的环境，它产生的黄曲霉毒素可以导致肝癌，严重地威胁着食品安全和人类健康。研究表明，黄曲霉毒素的产生受到多种信号途径的调控，但是磷酸化激酶slt2介导的毒素合成调控途径尚未见报道。本研究通过敲除黄曲霉的slt2基因，初步证实了该途径能显著影响黄曲霉的生长和产毒。为了进一步揭示slt2调控黄曲霉毒素产生的分子机制，本研究拟对slt2各个功能域进行突变，深入了解slt2起作用的具体部位；同时，对slt2调控的已知转录因子进行敲除，确定slt2调控的途径。最后，本研究还将通过EMSA和酵母单杂交技术确定转录因子作用到产毒基因上的具体序列。因此，该研究不但能够揭示磷酸化激酶slt2介导的信号途径在黄曲霉产毒中的生物学功能，还有望为控制黄曲霉毒素污染提供新的方法。

细胞丝裂原蛋白激酶途径（MAPK）在在许多病原真菌中都起着重要的作用。但是，在产黄曲霉毒素的黄曲霉中还没有报道。在本研究中，我们详细描述了MAPK中的一条途径MAPK－Slt2途径。通过定向删除AflSlt2基因，我们发现该激酶对于菌丝生长、孢子产生和菌核形成是必需的。对AflSlt2突变体的分析发现，该突变体对细胞壁损伤试剂极为敏感，但是对氧化胁迫表现出耐受增强。有趣的是，在培养基中，AflSlt2突变体比野生型能产生更多的黄曲霉毒素。不过，致病性实验发现突变体对花生的致病性降低。定点突变研究表明，AflSlt2的功能依赖于激活环内的磷酸化和亚结构域II中磷酸传递残基的磷酸化。综上所述，我们认为Slt2-MAPK途径参与了黄曲霉的发育、胁迫响应、毒素的产生和致病性。这为今后研制黄曲霉的防治措施奠定了基础。