Rab蛋白调控红曲霉中桔霉素合成的分子机制

Monascus spp are an important industrial fungi in China. The safety of Monascus fermented products is paid attention to owing to the health threats from the mycotoxin citrinin. In the synthesis of secondary metabolites, Rab GTPase is responsible for the transport of synthetic enzymes by regulating vesicle transport and providing a place for the synthesis of metabolites. In this study, analyzing expression levels of Rab protein and citrinin synthetic enzymes and citrinin content from Monascus by real-time fluorescence quantitative PCR and LC-MS analysis, candidate Rab genes related to synthesis of citrinin will be screened to study their functions. Colocalization of Rab protein and citrinin synthesis enzyme will determined by constructing them fused with the fluorescent protein and laser scanning confocal microscopy. We will compare transcriptome and proteomic changes through RNA-seq and iTRAQ mass spectrometry quantitative analysis of Rab gene overexpression or knock out mutant. The molecular mechanism of Rab GTPase regulation of citrinin biosynthesis will study. We will reveal the effect of correlation between Rab GTPase on citrinin biosynthesis and clarify the relationship between vesicles transport switch Rab GTPase and citrinin biosynthesis. The results will lay the foundation for the complex regulatory network of citrinin biosynthesis, and provide a theoretical reference for reducing citrinin pollution in Monascus fermented products.

红曲霉是我国重要的药食同源真菌，产生的真菌毒素--桔霉素影响了其安全性。在次级代谢产物的合成过程中，Rab小G蛋白通过调控囊泡运输负责合成酶的转运及为代谢物的合成提供场所。本项目拟通过实时荧光定量PCR和LC-MS分析红曲霉中Rab蛋白和桔霉素合成酶的转录水平及桔霉素含量，筛选与桔霉素合成相关的候选Rab基因进行功能研究，利用构建荧光蛋白融合蛋白与激光共聚焦对Rab蛋白和桔霉素合成蛋白进行共定位，通过RNA-seq和iTRAQ质谱定量分析比较Rab基因过表达或敲除菌株的转录组学和蛋白质组学变化，研究Rab蛋白调控桔霉素生物合成的分子机制，揭示Rab蛋白对桔霉素合成途径的相关性影响及其组分变化，阐明囊泡运输的分子开关Rab蛋白与桔霉素生物合成的相关性，为阐明桔霉素生物合成的复杂调控网络奠定基础及减少红曲及其制品中桔霉素污染提供一种思路和理论参考。

红曲菌是我国重要的药食同源真菌，产生的真菌毒素--桔霉素影响了其安全性。在次级代谢产物的合成过程中，Rab小G蛋白通过调控囊泡运输负责合成酶的转运及为代谢物的合成提供场所。本研究采用比较转录组学分析、基因敲除、荧光共定位等技术，对红曲菌Rab小G蛋白调控桔霉素合成的机制进行探索，通过比较转录组学分析，挖掘出与桔霉素合成相关的Rab基因，在9个Rab基因中，Rab6和Rab17明显与桔霉素的合成相关；通过将含融合目的基因和GFP或mcherry基因的载体转入红曲菌中，采用荧光共定位发现Rab6和Rab17蛋白定位于细胞内膜或内质网上，且与桔霉素合成酶能共定位于膜泡结构上，与转运蛋白共定位于细胞膜上；根据同源重组原理通过农杆菌介导的转化方法将打靶载体转入红曲菌中来构建Rab6和Rab17基因缺失株，经多轮单孢分离和PCR验证，发现转化子是异核体且生长状态明显受到影响，无法获得纯合子，说明Rab6和Rab17基因是生长所必需的。Rab6蛋白可能通过影响桔霉素合成途径中的调控因子ctnA和ctnR的表达水平进而影响桔霉素的合成。