沙糖桔青霉菌中桔青霉素生物合成关键基因的鉴定与功能研究

Citrinin is one of major mycotoxins, it can be kidney toxicity, as well as carcinogenic, teratogenic and mutagenic. Therefore, to clarify its biological formation and regulation mechanism is the basis for reducing mycotoxin contamination risk and ensuring food safety. Using the most serious diseases of sugar orange in postharvest storage, Penicillium citrinum as a model, and according to our previous work that pksCT was already cloned and sequenced from P. citrinum, which is a biosynthetic gene of citrinin. This project plans to explore the molecular mechanism of citrinin biosynthesis with the starting point from genes function and regulation. The main research contents include cloning gene cluster coding the biosynthesis of citrinin in P. citrinum, analyzing genes code region and genome structure, establishing a relationship between the genes expression and yield of citrinin. By using environmental factors, and the techniques such as RNA interference, high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR), it will verify or confirm that the function of key genes in intermediates formation during the process of citrinin biosynthesis, which is to clarify the ways of genes regulation. The expected results could preliminarily clarify the molecular regulation mechanism of citrinin biosynthesis in P. citrinum and the metabolites regulated by its key genes. At the meantime, it is useful in providing theoretical basis for the proposed new control strategies of citrinin contamination.

桔青霉素是主要真菌毒素之一，能引起人和动物的肾毒性， 致癌、致畸、致突变。因此，阐明其生物形成与调控机制是减少真菌毒素污染风险、确保食品安全的基础。本项目以沙糖桔果实采后贮藏病害中最为严重的青霉病菌为模式，在前期克隆了桔青霉素合成关键基因pksCT的基础上，从基因的功能和调控为切入点探索桔青霉素生物合成的分子机制。主要研究内容包括：柑桔青霉菌中桔青霉素生物合成基因簇的克隆、编码基因及基因组结构的分析，基因表达量与桔青霉素产量间关系的建立，通过环境因素控制、利用RNA干扰技术、结合高效液相色谱法、核磁共振等分析技术，验证或确定桔青霉素基因簇若干关键基因在其中间产物形成过程中的作用，进而阐明调控方式。研究预期成果可初步阐明柑桔青霉菌中桔青霉素生物合成的分子调控机制及其关键基因调控的代谢产物，同时为提出控制桔青霉素污染的新策略提供理论依据。

桔青霉素是主要真菌毒素之一，其生物合成关键基因的功能及调控途径是本项目的研究重点。本研究克隆了桔青霉菌中桔青霉素生物合成的关键基因pksCT，CtnR和CtnA等，揭示了其表达水平会受不同碳源的影响。尽管桔青霉素生物合成的分子路径已经比较清楚（Chem. Sci. 2016, 7, 2119–2127），但是我们的研究进一步发现其调控主要相关于氧化应激、初生和次生代谢、信号传导等。我们还发展了一种利用青海弧菌快速检测桔青霉素等其它真菌毒素毒性的方法，青海弧菌的发光强度与真菌毒素毒性大小之间的相关性R2>0.98，进一步我们测序了青海湖菌基因组，明确了其lux 发光操作元件。除此之外，我们对层生镰刀菌合成伏马毒素的调控机理展开了研究，证明了其合成受到BHA、pH值、碳源等影响。另外，我们的研究还阐明了桔青霉素和赭曲霉素的联合毒性导致人肾脏HEK293细胞凋亡及细胞周期捕获，其调控机制相关于抑制基因PDCD10和CCNG1的表达，并且分别上调了hsa-miR-1-3p和hsa-miR-122-5p。综上所述，我们的研究成果有望为降低真菌毒素污染的风险、确保水果安全的防治措施提供技术支撑。