氨氮胁迫下凡纳滨对虾血细胞的凋亡通路研究

Ammonia is a common environmental stress factor during the culture of shrimp. It is one of the main reasons for reduced immunity,disease and death of shrimp. Our previous study found that reactive oxygen species (ROS) production of haemocyte and haemocyte apoptosis ratio significantly increased in Litopenaeus vannamei exposed to ammonia. We hypothesize that shrimp may eliminate the serious damaged haemocyte by cell apoptosis. This process may be a protection strategy via physiological accommodation for ammonia stress. The present project will investigate the regulatory mechanism of haemocyte apoptosis in shrimp L. vannamei exposed to ammonia-N, according to our previous finding. Firstly, we will analyze the relationship between haemocyte apoptosis and ammonia dose/exposure time. Then we will detemine the occurrence of various apoptotic events by flow cytometry. On the other hand, the cDNA ibrary and digital gene expression library in haemocyte of ammonia-exposed shrimp will be constructed using the next generation sequecing technology RNA-seq. Then the genes with changed expression levels will be identified, and the expression levels of apoptosis-related genes will be analyzed. The differences of genes expression during the occurrence of apoptosis will be analyzed. The apoptosis pathway of haemocyte in ammonia-exposed L. vannamei will be demonstrated according to the results at cellular and molecular levels. The results of this study will have an important theoretic significance on deep research of anti-stress mechanism in shrimp. Moreover, the finding of the present study will have an important significance on the development of healthy aquaculture of shrimp.

氨氮是虾类养殖过程中最常见的环境胁迫因子，是造成虾类免疫功能低下、甚至大量发病和死亡的主要原因之一。本项目组前期的研究发现，氨氮胁迫下凡纳滨对虾血细胞的活性氧含量显著上升，随后血细胞发生显著的凋亡，我们推测虾类可能通过血细胞凋亡来清除受到过度氧化损伤的血细胞，这可能是虾类抗氨氮胁迫的生理调节保护策略。本项目拟针对此前期发现，深入研究氨氮胁迫下凡纳滨对虾血细胞凋亡的调控机制。先确定血细胞凋亡与氨氮浓度和胁迫时间的关系；然后应用流式细胞术分析血细胞各类凋亡事件的发生；另一方面，应用RNA高通量测序技术构建氨氮胁迫的转录组和表达谱文库，比对筛选差异基因并分析凋亡相关基因的表达变化，综合细胞和分子水平的研究结果阐明氨氮胁迫下血细胞的凋亡通路。本研究成果对深入研究虾类抗胁迫防御机理具有重要的理论意义，对虾类养殖业的健康发展具有重要的现实意义！

氨氮是虾类养殖过程中最常见的环境胁迫因子，是造成虾类免疫功能低下、大量发病和死亡的主要原因之一。前期研究发现氨氮胁迫会导致虾类血细胞凋亡。本项目针对此发现，深入研究氨氮胁迫下凡纳滨对虾的血细胞凋亡通路。先确定凋亡与氨氮浓度和胁迫时间的关系；然后分析血细胞各类凋亡事件的发生；构建氨氮胁迫的转录组和表达谱文库，比对筛选差异基因并分析凋亡相关基因的表达变化，综合细胞和分子水平的研究结果阐明氨氮胁迫下血细胞的凋亡通路。结果显示，胁迫24h为凋亡显著发生的时期，胁迫过程中相继发生活性氧（ROS）和一氧化氮（NO）的大量产生，磷脂酰丝氨酸外翻，线粒体膜电位（MMP）下降，胞浆Ca2+含量上升，caspase阳性率提高等凋亡事件；分子水平研究显示抗氧化酶、热激蛋白（HSP）及caspase3的表达量被不同程度地诱导；转录组与表达谱分析显示差异表达基因中包含大量外源性物质降解和代谢、DNA 修复、抗胁迫等相关基因；筛选出细胞色素c（Cyt C）、caspase 9、NADPH氧化酶和细胞色素P450（Cyt P450）进行了中间片段克隆，检测发现氨氮胁迫不同程度地诱导了这些基因的表达。结果表明，氨氮胁迫引起血氨含量上升，血氨作用于NADPH氧化酶及线粒体，导致胞内ROS含量大量上升，对线粒体、内质网等产生氧化胁迫；内质网受损导致内部Ca2+释放到胞浆中，激活一氧化氮合酶，导致NO产生，加重氧化胁迫；胞浆Ca2+会积累于线粒体，使线粒体膜受损，同时ROS与NO造成氧化损伤，导致MMP下降，细胞色素c释放到胞浆中，从而激活caspase9，继而激活caspase3及后续凋亡过程；氨氮胁迫还抑制了多种DNA修复蛋白的表达，是促使血细胞凋亡的重要因素之一。抗氧化酶、HSP、Cyt P450的表达上调表明它们在抗氨氮防御中发挥重要作用。通过本项目研究，初步明确了氨氮胁迫下凡纳滨对虾血细胞的凋亡路径，同时挖掘了一些抗氨氮胁迫相关因子，为进一步研究如何提高虾类的抗氨氮胁迫能力提供了研究基础，对深入研究虾类抗胁迫防御机理具有重要的理论意义，对虾类养殖业的健康发展具有重要的现实意义！