SDR蛋白参与胶红酵母降解苹果展青霉素的分子机制研究

Patulin is one of the mycotoxins that can cause serious health problems, which has been identified in apple fruit and its products throughout the world. Controlling and degradation of patulin on apple fruit and its products using antagonistic yeasts has represented a promising future. The applicant had studied the control of patulin in apples by Rhodotorula mucilaginosm and it's response and regulation mechanisms of degradation of patulin, the differentially expressed proteins and differentially expressed genes related to patulin degradation by R. mucilaginosa were analyzed through an iTRAQ-based proteomic analysis and RNA-Sequencing technology, short-chain dehydrogenase (SDR protein) in R. mucilaginosa treated with PAT was significantly up-regulated expressed. It is indicated that SDR protein play an important role in patulin degradation by R. mucilaginosa, but the molecular mechanism is not clear. In this study, SDR protein is selected to be the model object, the expression of SDR gene and SDR protein which are stimulated by patulin will be determined. SDR gene knockout mutants and overexpression strains of R. mucilaginosa will be constructed to prove the efficacy in patulin degradation. The catalytic mechanism of SDR protein on patulin was analyzed by heterologous expression and purification of SDR. The transcription factor of SDR and its regulation mechanism will be studied, so as to reveal the mechanisms of SDR protein in degrading patulin by R. mucilaginosa. The expected research results will provide theoretical evidence for the application of antagonistic yeasts to degrade PAT of apple fruit and its products.

展青霉素（PAT）是对人类危害最大的真菌毒素之一，其对苹果及其制品的污染在全球范围内都很严重。采用拮抗酵母控制并降解苹果及其制品中的PAT已展现出良好的应用前景。申请者前期研究胶红酵母控制苹果PAT及降解的应答调控机制，利用iTRAQ技术和转录组测序技术分析其降解PAT的差异表达蛋白和差异表达基因时，发现PAT处理的酵母中短链脱氢酶（SDR蛋白）的表达水平显著上调，说明SDR在胶红酵母降解PAT的过程中发挥着重要作用，但其参与PAT降解的分子机制未知。本项目拟以SDR蛋白为研究对象，测定SDR基因和蛋白在PAT刺激下的表达水平；构建SDR敲除及过量表达菌株，分析其在酵母降解PAT中的作用；外源表达并纯化SDR，分析其对PAT的催化机制；对调控SDR的转录因子及其调控机制进行研究，从而阐明SDR参与胶红酵母降解PAT的分子机制。研究成果可为拮抗酵母降解苹果及其制品中PAT的机制提供理论支撑。

展青霉素（Patulin，PAT）是一种有较强毒性的聚酮类次级代谢产物，主要由扩展青霉产生，其侵染采后水果会造成腐败，并在侵染过程中产生PAT。随着对水果采后病害进行生物控制研究的进展，利用拮抗酵母控制并降解水果及其制品中的PAT已经成为学术界研究的热点。课题组筛选到一株季也蒙毕赤酵母（Meyerozyma guilliermondii），前期研究发现M. guilliermondii可显著抑制梨果上PAT的积累，且M. guilliermondii能体外降解PAT。但是，M. guilliermondii降解PAT的分子机制仍然未知，这限制了其实际应用。本项目针对这一问题进行研究，研究内容主要包括：基于转录组学技术研究M. guilliermondii在基因表达水平上对PAT的应答调控机制；确定PAT刺激M. guilliermondii后参与其降解PAT的关键基因MgSDR；构建MgSDR的过表达菌株，验证MgSDR参与PAT降解过程的功能；构建MgSDR的外源表达菌株，初步研究并确定了MgSDR酶对PAT的降解作用；基于染色质开放性测序技术研究了M. guilliermondii在表观水平上对PAT的应答调控机制；通过酵母单杂交筛选到4个潜在调控MgSDR的转录因子，并对筛选到的转录因子进行了亚细胞定位。转录组学分析表明，PAT刺激提高了与耐药、抗氧化胁迫等防御相关基因的表达，帮助M. guilliermondii抵御PAT毒性；转运蛋白相关基因上调表达，可能参与到PAT转运中；与PAT降解相关的SDR基因上调表达，推测其在PAT降解过程中发挥重要作用。染色质开放性测序表明PAT胁迫影响了类固醇生物合成、ABC转运蛋白、自噬酵母、mRNA监控途径、MAPK信号途径和M. guilliermondii内质网中的蛋白质加工。MgSDR基因过表达菌株对PAT的降解作用显著增强；外源表达MgSDR的大肠杆菌对PAT的降解作用显著增强。通过酵母单杂交筛选到了四个潜在的调控MgSDR的转录因子，其亚细胞定位主要位于细胞核及细胞质。项目的研究结果为寻找靶向降解PAT的酶及其编码基因提供了重要信息，后续可通过基因工程技术对该酶进行大规模生产，进而将其用于食品中PAT的清除。项目的研究结果具有重要的理论价值、应用价值和社会效益。