组蛋白去乙酰化酶介导的红曲菌次级代谢转录调控机制研究

Histones are highly alkaline proteins in nucleus of eucaryotic cell, acting as spools around which DNA winds. Histone acetylation modification modulates the transcription of gene clusters coding for fungal secondary metabolites by histone deacetylases, but the modulation mechanisms are of diverse. Monascus spp. are very important resources used both in the industry of food and medicine due to the production of various secondary metabolites. Our previous experiments revealed that different HDACs had diverse effects on the transcription level of secondary metabolites gene clusters and the production of secondary metabolites in Monascus ruber M7, but the regulation mechanisms are still not understood. In this project, these approaches including Western blot, ChIP-Seq, ChIP-qPCR ,Co-IP and MS will be used to investigate the modulation mechanisms on Monascus secondary metabolites gene clusters mediated by HDACs at the level of histone–DNA binding, and interaction of protein-protein, which will lay foundation for modulating the production of Monascus secondary metabolites and enriching the modulation theory of HDACs on fungal secondary metabolites.

组蛋白是真核细胞核中与DNA结合的碱性蛋白总称。组蛋白去乙酰化酶（histone deacetylases，HDACs）介导的去乙酰化程度调控着真菌次级代谢基因簇（secondary metabolites gene cluster，SMGC）的转录，但机制存在多样性。红曲菌（Monascus spp.）是我国重要药食同源真菌，可分泌多种有益SM，应用前景广阔。前期研究表明，红色红曲菌M7中HDACs对SMGC转录及SM产率影响不同，但机制不清楚。本项目拟以红色红曲菌M7的HDACs缺失株为材料，原始菌株M7为对照，采用Western blot、ChIP-Seq、ChIP-qPCR和 Co-IP等方法结合质谱技术从组蛋白-DNA、蛋白互作等方面研究HDACs对红曲菌SMGC转录调控机制。研究结果不仅为调控红曲菌次级代谢产物生产提供理论指导，也可丰富HDACs对真菌SMGC调控机制。

组蛋白去乙酰化酶（histone deacetylases, HDACs）在真菌代谢和发育中具有重要的、多样化的调控作用，前期研究发现红色红曲菌中三个命名为MrHda1、 MrHos2 和 MrSir2的HDACs对红色红曲菌中两种主要的次级代谢产物——红曲色素和桔霉素产率具有显著的调控差异。为解析这种调控差异的机制，本项目从HDACs调控的基因转录及蛋白的翻译后修饰两个层次进行研究。转录组数据分析显示在不同生长阶段，MrHda1、MrHos2和MrSir2能够调控色素和桔霉素基因簇中基因表达的数目和调控作用各不同，并且调控基因的表达变化趋势与代谢产物的变化趋势是一致的，表明MrHda1、MrHos2和MrSir2在转录水平对色素和桔霉素合成基因簇存在调控的差异。Western blot结果显示，MrHda1、MrHos2和MrSir2分别缺失后，菌株总蛋白乙酰化修饰程度分别出现了上升、无变化和显著上升的趋势；对组蛋白不同亚基上赖氨酸残基乙酰化修饰既存在一致性，也存在差异性，其中MrSir2主要参与早期生长过程中组蛋白亚基赖氨酸乙酰化修饰作用。鉴于H3K56具有驱使染色质转变成转录激活过程中分解状态的功能，采用ChIP-qPCR技术检测三个HDACs缺失后参与色素和桔霉素合成的聚酮合酶和调节基因启动子结合区域的乙酰化修饰变化。结果显示H3K56修饰在ΔMrHda1和ΔMrSir2菌株检测位点处略有下降，在ΔMrHos2菌株中显著上升。综合以上结果，推断MrHda1、MrHos2和MrSir2主要从调控色素和桔霉素基因簇转录的水平调控产物的合成。研究结果丰富了HDACs在红曲色素和桔霉素合成过程中的调控作用机制，也丰富了真菌次级代谢的调控理论。