氮代谢调控因子AaAreA调控AOH毒素生物合成的分子机制

Alternariol (AOH) is a mycotoxin produced by Alternaria species, which mainly contaminates grains, fruits and vegetables. It exhibits mutagenicity and potential carcinogenicity. In the previous study, nitrogen sources could inhibit AOH biosynthesis as an important signal. Unlike the other mycotoxin producing fungi, various nitrogen sources (ammonium, nitrate, and arginine) suppressed the AOH production in Alternaria. AaAreA, as a key transcription factor for nitrogen metabolism regulation, have been identified to regulate AOH biosynthesis. However, it is unclear about molecular mechanism of this regulation. Therefore, in this research, based on wild Alternaria strain, the obtained AaAreA gene knockout mutants, and the reverse mutants that would like to construct, we will compare the changes of AOH production, growth and sporulation. We will identify the dynamic distribution of AaAreA in Alternaria cell under different nitrogen sources by constructing AaAreA-GFP fusion protein. Then, we will uncover the regulatory pattern of AaAreA on controlling AOH production by exploring the interaction of the heterologously expressed AaAreA protein and the specific regulatory gene AaAohR in AOH biosynthetic gene cluster, and the gene function of AaAohR. Finally, we will systematically illuminate the regulatory network of AaAreA on controlling AOH production under different nitrogen sources in combination with transcriptomic analysis. The study may provide new insights and scientific basis for the potential application of green and safe nitrogen sources inhibiting the mycotoxin biosynthesis.

交链孢酚（AOH）是主要由交链孢菌产生的真菌毒素，广泛污染谷物和果蔬，具有致突变和潜在致癌性。氮源作为重要信号影响真菌毒素合成。与其他产毒真菌不同，不同氮源（铵盐、硝酸盐和精氨酸等）均抑制交链孢产AOH，且氮代谢调控因子AaAreA可调控AOH合成，然其分子机制尚不明确。因此，本研究利用交链孢野生型、已获得的AaAreA敲除突变株及拟构建的回复突变株，比较不同氮源条件菌株产AOH及生长产孢变化；构建AaAreA-GFP融合蛋白，明确不同氮源下AaAreA在细胞中的动态分布；通过异源表达获得AaAreA蛋白，研究该蛋白与AOH合成基因簇内调控基因AaAohR的互作以及明晰AaAohR的基因功能，揭示AaAreA调控AOH合成的作用方式；结合转录组学系统分析AaAreA对AOH合成的调控网络，最终阐明AaAreA调控AOH合成的分子机制，以期为利用绿色安全氮源抑制毒素合成提供新思路和科学依据。

互隔交链孢是重要的植物病原菌，且可产生交链孢酚（AOH）等真菌毒素，造成粮食和果蔬污染。AOH具有致突变、致畸和潜在致癌性，严重危害人体健康。氮源作为营养因子可影响AOH毒素合成，而AaAreA是氮代谢调控的关键转录因子。为解析AaAreA对AOH合成的调控机制，本研究分析了不同氮源对互隔交链孢菌株产毒能力的影响，发现次级氮源NaNO3较之初级氮源NH4Cl更加有利于AOH毒素累积；明确了不同氮源下AaAreA与AOH合成的关系，结果显示AaAreA可正调控AOH毒素的合成；分析了不同氮源下AaAreA调控的时空关系，表明细胞利用次级氮源时，AaAreA经转运于细胞核富集，调控硝酸盐还原酶等基因表达，进而将NO3-还原为便于利用的NH4+，保障互隔交链孢各种生命活动的正常运行；进一步研究了AaAreA与AOH合成基因簇内转录因子基因AaAohR（aohR）的互作关系，证实了AaAreA可与aohR启动子序列（5’-GGCTATGGAAA-3’）结合，进而激活其转录，而其表达蛋白可调控AOH合成簇内合成酶基因表达水平，最终影响AOH毒素合成；从转录组角度更进一步系统揭示了不同氮源下AaAreA对AOH合成的调控网络。该研究为氮肥施用的选择、绿色安全的氮素抑制毒素合成的潜在应用提供了新的思路和理论依据。