一氧化氮在对虾免疫应答中的角色及调控

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.

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。

采用RNAi技术及NO供体，解析了NOS表达受到抑制及激活状态下，NO产生被抑制及大量激活后，对对虾免疫功能的影响；克隆了与NO调控密切相关的GCIII、精氨酸酶、CYC、AIF、ATG5与LC3的全长cDNA，采用qPCR对其组织及WSSV刺激后的时空表达谱、细胞凋亡进行了分析；分析了NO合成受到抑制及激活状态下，NO合成、cGMP含量、基因表达与WSSV增殖之间的关系；查明了温度胁迫、WSSV感染与对虾ROS产生、抗氧化酶变化、能量产生之间的关系；构建了一个对虾的T7噬菌体展示文库，对NOS进行了原核表达，并在文库中淘选了与NOS相互作用的蛋白；采用代谢组学分析了NO合成受到抑制/激活状态下，WSSV感染后，对虾代谢模式变化。.结果表明：敲低NOS，THC降低，NO合成减少，WSSV增殖增加；供体诱导NO合成增加，WSSV增殖受阻。GCIII基因编码284 aa，遍布于对虾多组织，病毒感染诱导其表达上调、cGMP含量增加；敲低NOS，GCⅢ和cGMP及WSSV增殖增加。凋亡因子CYC、AIF分别编码104与440个aa，病毒可以诱导肝胰腺中CYC与AIF上调，caspase3增加，细胞凋亡；而血细胞凋亡在病毒感染早期被抑制，推测WSSV可能在感染初期通过抑制血细胞凋亡实现自我复制与感染。自噬因子ATG5与LC3分别编码269与146个aa，病毒感染抑制ATG5和LC3表达，细胞自噬清除病毒过程受损。精氨酸酶基因编码 332个aa，病毒可以抑制其转录，使对虾精氨酸/NO代谢途径受损，实现增殖。病毒及温度胁迫导致机体ROS增加，抗氧化酶合成增加，能量消耗增多，提示在温度胁迫后，对虾需要更多能量维持机体平衡，抵抗病原感染。原始T7噬菌体展示库重组率97%，大于250bp片段为70%，滴度9×107。获得NOS重组蛋白，利用T7噬菌体展示库，淘选到COXIII与EF1A可能与NOS互做，提示NO调控与线粒体可能密切相关。NO合成被抑制及激活后，病毒感染引起对虾代谢模式变化。本研究为深入理解NO在对虾抵抗WSSV感染中的作用奠定基础。