红鱼干中镰孢菌TORC1信号系统介导的氨基酸对T-2毒素合成的调控机制

Dried Lutianus erythropterusis an aquatic specialty in the South China Sea. During its production and storage period, the development of free amino acid (FAA) and T-2 toxin (T-2) contamination are closely related. It is therefore helpful to find out FAA’s regulation mechanism of T-2 synthesis in order to precisely control the quality and safety of dried Lutianus erythropterus. In this project, ∑FAA will be extracted from the dried Lutianus erythropterus samples taken in the production and storage period and will then be added into the substrates of Fusarium Fo17. LC-MS/MS method will be used to analyze the yield of T-2; Real time PCR will be used to analyze TRI5 expression quantity; and phenotypic characteristics will be observed according to morphology. As such, the key FAA (FAAk) relation to T-2 synthesis will be found and its standards can be used to perform T-2 producing verification. In the meantime, homologous recombination method will be used to construe null mutant strains of the upstream response elements (Gtr1 and Gtr2) and the downstream T-2 regulatoryelements (SCH9 and TAP42) in TORC1 signal system respectively, and to analyze the function of each element in the process of T-2 synthesis and TRI5 expression in order to ascertain the key upstream and downstream genes that are related to FAAk. The function of the key genes will be verified through gene recovering technology. Finally, immune affinity capture and precipitation method will be adopted to discover the effect of TORC1 protein molecule on the formationof upstream and downstream target element complex, so as to illustrate the cascade regulatory mechanism of T-2 synthesis induced by FAAk.

红鱼干作为我国南海特色水产珍品，加工贮藏过程中游离氨基酸（FAA）变化与镰孢菌T-2毒素污染密切相关，因此，探明FAA调控T-2合成的机制有助于红鱼干质量安全的精准控制。本项目提取红鱼干加工贮藏期样本的∑FAA，添加到镰孢菌Fo17 的培养基中，采用LC-MS/MS分析T-2产量、Real time PCR分析TRI5表达量、形态学表征镰孢菌，找出T-2合成的关键FAA（FAAk）并以其标准品进行产毒验证。同时，利用同源重组技术分别构建TORC1信号系统上游的FAAk响应元件（GTR1和GTR2）和下游的毒素调控开关（SCH9和TAP42）缺失株，分析各元件在T-2合成和TRI5表达过程中的作用，明确与FAAk相关的上游和下游关键元件，并以基因回补技术验证其功能。最后以免疫亲和捕获和沉淀技术解析TORC1蛋白分子上下游目标元件复合体形成中的功能，进而阐明FAAk对T-2合成的级联调控机制。

红鱼干作为一类高蛋白低水分水产干制品，在加工贮藏过程中极易受到镰孢菌污染，产生真菌毒素，对食品安全造成严重威胁。鱼干在长期贮藏过程中，蛋白质分解形成游离氨基酸，作为营养物质被真菌利用于生长繁殖。本项目研究FAA 对鱼干源产毒镰孢菌的生长及代谢的作用机制，深入解析TORC1信号通路介导镰孢菌对特征氨基酸的响应和对生长代谢及生物合成的调控机制。利用同源重组双交换法，经感受态细胞转化、质粒DNA提取及原生质体制备、构项目项项目建基因敲除载体、转化子筛选及PCR验证，获得△FoGtr1、△FoGtr2、△FoSch9、△FoTap42基因敲除株；经构建回补载体、农杆菌转化、农杆菌-真菌共培养、转化体筛选及PCR验证，得到基因回补株。研究表明中/碱性氨基酸如甘氨酸（Gly）、L-His、L-Pro和L-Thr激活镰孢菌生长；酸性氨基酸如天冬氨酸（L-Asp）、L-Glu和含硫氨基酸L-Cys、L-Met抑制镰孢菌生长。L-Thr和L-His可显著提高镰孢菌野生株、△FoTap42和△FoTap42-C产毒能力，且呈高剂量激活效应，但Tap42缺失使Tri5基因表达降低；低剂量L-Asp显著激活镰孢菌产毒能力，随剂量升高T-2合成和Tri5基因表达显著被抑制。经构建pET28a(+) -Tap42表达载体，蛋白原核表达及纯化，联合质谱分析，得到与Tap42相互作用的蛋白有：DNA拓扑异构酶2、3-磷酸甘油醛脱氢酶、70kDa热休克蛋白 、翻译延长因子EF-1α、肌动蛋白、质膜ATP酶、烯醇酶、ATP合酶亚基、转醛醇酶和肽链延伸因子 2等，关联的调控代谢途径主要来自于新陈代谢、遗传信息处理、细胞转化和环境信息处理。本研究结果表明，氨基酸对TORC1信号通路介导尖孢镰孢菌生长、产孢能力和毒素合成等重要生命活动具有显著调控作用。研究结果为后续靶向防控镰孢菌在鱼干中的生长和产毒提供理论基础。