一氧化氮（NO）由一氧化氮合酶（NOS）催化L-精氨酸生成，在对虾免疫防御中具有重要作用。同时，对虾体内L-精氨酸与ATP浓度均受精氨酸激酶（AK）调控，NO合成、对虾免疫力与AK催化的ATP平衡还与温度胁迫密切相关。因此，本项目拟采用RNAi及抑制剂使NOS表达/活性缺失，检测免疫刺激后对虾NO含量、相关免疫指标、病原增殖、对虾死亡率等变化，解析NO在对虾免疫反应中的角色；通过分析免疫刺激、温度胁迫及NOS底物、辅酶等对NO合成/NOS表达、ATP合成/AK表达及对虾免疫力之间的关系，采用蛋白质组学分析不同刺激/胁迫交互作用中NO合成及调控关键点蛋白质表达变化，阐释NO合成及调控；对NOS重组表达，制备抗体，细胞定位，采用蛋白下拉及酵母双杂交等方法鉴定并验证与NOS相互作用蛋白质，解析NO合成调控的蛋白网络，丰富对虾免疫学知识，并为气候变化下的对虾健康养殖提供理论依据。

Nitric oxide (NO) is known as one of the most versatile players in animals' immune system. It is also known as one of the crucial immune effectors that involve in the pathogenesis and infectious diseases controling in shrimp. NO is a product of L-arginine conversion to L-citrulline by nitric oxide synthase (NOS) enzyme. In shrimp, the production of L-arginine and ATP are catalyzed by arginine kinase (AK). More important, the regulation of NO synthesis, the immune response and the energy metabolism in shrimp show a close correlation with environmental temperature. Therefore, we first want to know the role of NO in shrimp immune response through detecting the change of NO concentration, NOS expression and some important related immune parameters after NOS silence by RNA interference or activity inhibition by its inhibitor. Then, we intend to clarify the relationship among NO synthesis, related immune process, pathogens reproduction and energy metabolism in shrimp after immune challenge, temperature stress and NOS regulator (such as L-Arginine and NADPH etc.) by biochemical assay, realtime RT-PCR, westernblot and cytology methods. The relationship and interaction among these precess and biological events and their interaction will be analyzed and the most important samples for NO synthesis after different challenge will be further investigated by proteomic method to reveal its regulation mechanism. Third, the NOS will be recombinated and expressed in vitro, its antibody will be prepared as well as the localization of NOS in cells will be done. Moreover, identification and verification of the interaction protein with NOS by GST-pulldown and yeast two hybrid or Co-IP and the protein that interact with NOS will be uncovered. Our results will bring some new knowledge in shrimp immunology area and provide some important data for shrimp healthy culture under the changing climate.