氨氮胁迫下凡纳滨对虾CHH调控糖代谢信号通路的机制研究

Changes in glycogen and total carbohydrate concentrations induced by stress are accompanied by an increased secretion of CHH in shrimps. However, downstream signal pathways of CHH that mediate these processes remain unknown. Ammonia is one of the major environmental stressor, influencing the growth and survival of shrimps seriously. Thus it has a great theoretical value to clarify the regulatory mechanisms of endocrine hormone on carbohydrate metabolism signal pathway in response of ammonia. In this study, we plan to understand how CHH mediates carbohydrate metabolism signal pathways and affects carbohydrate metabolic enzymes, illuminate the regulatory mechanisms of CHH on signal pathways and carbohydrate metabolism enzymes in response of ammonia in Litopeneaus vannamei. Firstly, detect whether CHHBP is able to interact with CHH and the downstream signal pathway of CHHBP. Then, investigate the effects of CHH on carbohydrate metabolism signal pathway and metabolic enzyme by in vivo and in vitro methods. Finally, study the temporal expression profiles of CHH, signal genes, carbohydrate metabolic enzyme under ammonia stress. This project will provide theoretical basis for molecular mechanism on carbohydrate metabolism regulation of CHH in Litopeneaus vannamei, and provide new academic information to the tolerance and adaptation mechanisms induced by the environmental ammonia in Litopeneaus vannamei.

虾类受到胁迫会释放内分泌激素CHH引起糖代谢紊乱，而CHH对虾肝脏糖代谢信号通路的作用机制一直未能确切阐明。而氨氮作为养殖水体的重要胁迫因子，解析内分泌激素介导的糖代谢信号通路对氨氮胁迫的响应机制具有重要的理论价值。本项目拟以凡纳滨对虾为研究对象，开展CHH介导糖代谢信号通路及糖代谢酶活性调控研究，解析氨氮胁迫诱导的糖代谢变化和分子适应机制：首先，验证CHH和CHH受体互作关系以及鉴定下游信号通路；其次，通过体内和体外的验证方法，分析CHH对糖代谢相关通路基因、糖代谢酶等的影响，阐明CHH在糖代谢调控网络中的作用；最后，查明CHH蛋白介导的糖代谢通路对氨氮胁迫的响应。研究结果将解析CHH对凡纳滨对虾肝脏糖代谢信号通路的调控机制，并从内分泌激素、信号通路对糖代谢调控等角度研究氨氮对凡纳滨对虾的影响，为进一步了解凡纳滨对虾对氨氮的耐受机制提供理论基础。

糖代谢变化是甲壳动物在遭遇胁迫时的一种常见且敏感的生理效应，探索糖代谢的调控机制，是阐明胁迫诱导的糖代谢变化和分子适应机制的关键所在，其中甲壳动物高血糖激素CHH在血糖调节过程中发挥至关重要的作用。而氨氮作为养殖水体的重要胁迫因子，解析内分泌激素介导的糖代谢信号通路对氨氮胁迫的响应机制具有重要的理论价值。本项目主要成果为：1）筛选了凡纳滨对虾CHH和可能的受体基因，采用分子生物学技术分析CHH和flotllin基因序列和结构，采样酵母双杂，免疫荧光，RNA干扰等技术证实Flot-1和CHH6有相互作用；2）采用RNA干扰和注射CHH重组蛋白技术，分析CHH对凡纳滨对虾糖代谢相关通路关键基因（PI3K、Akt），糖代谢相关酶（HK、PK、PFK、LDH等）活性和表达水平，以及糖代谢相关底物含量的影响，分析CHH可以通过调控糖代谢PI3K-Akt信号通路来调节糖代谢关键酶，最终影响了糖代谢的产物如血糖和乳酸含量；3）采用RT-PCR等技术检测急性氨氮胁迫后，CHH水平显著升高，糖代谢通路基因（PI3K、Akt）水平显著下降，通过干扰技术结合氨氮胁迫，分析CHH6对代谢酶的影响，解析了CHH通过介导的糖代谢信号通路调节糖代谢的关键酶来响应急性氨氮胁迫。本研究结从内分泌激素、信号通路对糖代谢调控等角度研究氨氮对凡纳滨对虾的影响，解析CHH对凡纳滨对虾肝脏糖代谢信号通路的调控机制，为进一步了解凡纳滨对虾对氨氮的耐受机制提供理论基础。