环境胁迫下凡纳滨对虾加氧酶参与免疫应答的作用机制

As a key protein which can response to oxidative damage, oxygenase plays an important biological role on organism. Litopenaeus vannamei is the biggest variety of shrimp in the aquaculture industry of China, however, at present there is no oxygenase-related research on the shrimp. For the sake of discussing the mechanism of oxygenase involved in immunity regulation in the shrimp, this project makes an exhaustive study by the view of oxygenase function..Firstly we clone oxygenase from the shrimp, analyse its bioinformatics, confirm the protein expression, tissue localization, biochemical and cytological effect. Then we explore the transcription and translation regulation patter of oxygenase when the shrimp exposed to different environmental stress. In addition, by the application of RNA interference (RNAi) technology, we investigate the effect of oxygenase on cellular morphology, cell apoptosis and damage, regulation of cellular immunity and the relationship with other immune related factors. .This study aims to describe a new mechanism of how oxygenase participate in the immune regulation of Litopenaeus vannamei, which will provide a possible way to enhance the ability of resistance in shrimp under environmental stress.

加氧酶作为一种应答氧化损伤的关键蛋白，对机体发挥着重要的生物学作用。凡纳滨对虾是我国对虾养殖产业的最大优势品种，目前却尚未有关加氧酶的研究。本项目尝试从加氧酶的功能切入，深入研究加氧酶参与凡纳滨对虾免疫调控的机理。我们首先对加氧酶进行基因克隆及生物信息学分析，确定其蛋白表达、组织定位、生化活性和细胞学作用。继而分析凡纳滨对虾在各种不同环境胁迫下加氧酶基因、蛋白质表达谱及其活性。在此基础上我们利用RNA干扰技术探讨加氧酶对细胞形态、细胞凋亡和损伤、细胞免疫的调控作用及与其他免疫相关因子的关系。该研究旨在阐述加氧酶参与凡纳滨对虾免疫调控的崭新机理，为利用虾类自身的免疫调节基因加强抵抗外界环境胁迫的能力提供了可能途径。

凡纳滨对虾是我国对虾养殖产业的最大优势品种，目前尚未有关加氧酶的研究。为探讨环境胁迫下凡纳滨对虾加氧酶参与免疫应答的作用机制，在本项目中，首先利用RACE技术首次在凡纳滨对虾中克隆得到加氧酶cDNA全序列全长1383bp，编码39.6kD蛋白，通过构建表达载体成功表达加氧酶蛋白并制备了高效专一的多克隆抗体；然后通过实时定量、免疫杂交以及活性分析揭示了凡纳滨对虾在多种环境胁迫下加氧酶基因和蛋白质的时空表达诱导模式，表明加氧酶参与了凡纳滨对虾环境胁迫下的防御作用；随后利用RNA干扰技术抑制加氧酶表达，发现机体免疫系统受到攻击，细胞产生毒害作用并引起DNA损伤；进一步机制研究表明，加氧酶可以通过机体抗氧化酶系统及能量代谢系统相互协同作用。此课题对阐明环境胁迫下凡纳滨对虾加氧酶参与免疫应答的作用机制具有重要意义，为加氧酶作为水产动物环境胁迫标记物提供了可能。