对虾白斑综合症病毒（WSSV）蛋白对对虾免疫相关基因表达的调控

项目名称： 对虾白斑综合症病毒（WSSV）蛋白对对虾免疫相关基因表达的调控

项目编号： No.30800857

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 王蔚

作者单位： 国家海洋局第三海洋研究所

项目金额： 22万元

中文摘要： 对虾是我国海水养殖的主要对象，但是深受WSSV等病原的巨大威胁,至今没有有效的防治方法。本项目从对虾的四个重要免疫相关基因Rab和Ran、Caspase以及QM出发，研究WSSV病毒蛋白对对虾免疫应答的调控。首先采用染色体步行的方法，获得Rab和Ran、Caspase和QM基因的5'端上游序列，长度分别为180bp、827bp、678bp和307bp。将获得的序列在昆虫细胞中验证其启动子功能，发现Ran和Caspase的上游序列有启动子功能。将Ran和Caspase的调控区DNA进行亲和素标记，然后与链霉亲和素修饰的磁珠结合，通过下拉发现Ran基因的启动子序列与对虾的Actin和Ran蛋白结合，Caspase的启动子序列能与病毒的VP38 和VP41B蛋白结合。通过荧光素酶报告基因发现VP38和VP41B对PjCaspase启动子分别有抑制和激活作用，而对VP38和VP41B基因进行过量表达和RNAi抑制后，发现WSSV感染后这两个WSSV蛋白对对虾血细胞凋亡有调控作用。本项目研究结果为无脊椎动物的免疫机制增添了新的内容，也为对虾病害防治提供了基础。

中文关键词： 对虾；无脊椎动物免疫; WSSV; 转录调控

英文摘要： White spot syndrome virus (WSSV) is the major pathogen of shrimp in worldwide shrimp culture. However no efficient approach is available to control this virus. In this investingation, the molecular mechanism of shrimp antivirus is conducted based on the regulation of four immune related gene of shrimp(Rab, Ran, Caspase and QM). Through genome walking the 5'-flanking sequence of Rab, Ran, Caspase and QM are obtained. By fusing the 5'-flanking promoter region of the PjCaspase gene to the enhanced green fluorescence protein (EGFP) gene and transformed to Trichoplusia ni High Five_ cell line. By fusing the 5'-flanking region to the enhanced green fluorescence protein (EGFP) gene and transformed to Trichoplusia ni High Five cell line, the promoter activity of Ran and Caspase were confirmed. With streptavidin-bead pulldown assay, two envelope proteins VP38 and VP41B of white spot syndrome virus (WSSV) were found to bind to Caspase promoter, two shrimp proteins Actin and Ran were found to bind to Ran promoter. Luciferase reporter assay by cotransfection of PjCaspace promoter with VP38 or VP41B revealed that the proteins act as repressor and activator of PjCaspase transcription respectively. Overexpression and RNAi inhibition of VP38 or VP41B revealed the two WSSV proteins play important role in apoptosis. Our study provides novel insights into innate immunity of invertebrates and potential approaches for virus control in shrimp.

英文关键词： Shrimp; Invertebrate immunity; WSSV; transcriptional regulation