"类免疫"保护对中国对虾产生WSSV抗性的作用及其分子基础

The "Huanghai No. 2" is the new culture variety of Chinese shrimp (Fenneropenaeus chinensis) which was obtained after nine-generation successive selective breeding. In its WSSV challenge test, it showed high WSSV-resistance ability significantly that it can tolerate less than 10,000 WSSV copies. Besides this, it's found that the survival rate was improved significantly when they were challenged by WSSV once more, seemed like this new variety showed special function called "quasi-immune protection". We are wondering that if the WSSV-resistance ability of "Huanghai No. 2" was generated from the heredity alone? Or incorporated with "quasi-immune protection"? And can "quasi-immune protection" function be inherited? The present study was supposed to compare the WSSV-resistance ability and the difference of "quasi-immune protection" when challenged and re-challenged by WSSV using the two populations of "Huanghai No. 2" and nature population without selection background, as well as the filial generation of "quasi-immune" survivals next year; and to research the effects of WSSV-resistance function generated from "quasi-immune protection". Furthermore, in order to investigate the molecular basis of "quasi-immune protection" in a deep-going way, association analysis will be conducted using SNP markers and gene chips respectively which have been achieved by our study in prior period. The results of the present study will illuminate the effects and mechanism of "quasi-immune protection" in WSSV resistance in F. chinensis for the first time, which will provide the theoretical foundation for combining the "quasi-immune protection" with selective breeding and will improve the WSSV-resistance ability further. The concept in the present study will be a good demonstration for researchers when studying the disease resistance in other shrimps and crabs.

"黄海2号"是经连续9代选育的中国对虾养殖新品种，在抗WSSV性能测试中，发现其对WSSV有显著抗性（可耐受低于1万WSSV拷贝的病毒量）；也同时发现重复感染WSSV的存活率显著提高，似乎有"类免疫"保护功能。"黄海2号"抗性究竟单纯源于遗传，还是掺有"类免疫"保护作用？"类免疫"可否遗传？本课题拟以连续选育的"黄海2号"和未经选育的野生对虾为研究对象，通过比较两者（以及次年开始产生的"类免疫"子代）耐受WSSV能力和在二次人工感染后"类免疫"保护差异，研究"类免疫"保护对中国对虾产生WSSV抗性的作用；同时利用课题组前期研究已获得的SNP标记和基因芯片分别进行关联分析，深入探讨"类免疫"保护的分子基础。研究结果将首次阐释"类免疫"保护在对虾产生WSSV抗性中的作用和机理，为其与良种选育有效结合、进一步提高中国对虾抗病性提供理论依据。本研究思路还将对其他虾、蟹类抗病研究起到良好示范作用。

本项目以中国对虾“黄海2号”为研究对象，进行对虾耐受WSSV差异研究，发现只有当病毒粒子的含量高于104copies/mg时，才能引起白斑综合症的暴发。将含不同WSSV拷贝的病毒饲喂对虾后，感染30天后的对虾存活率差别较大。二次感染后，各实验组的存活率之间没有差异，但是初次感染后的存活个体再次感染时，各组的平均存活时间则差异显著（P<0.05）。使用热灭活的白斑综合征病毒投喂不同水温条件的实验组对虾，发现热灭活WSSV可诱导对虾产生免疫致敏反应，对受WSSV感染的对虾具有一定的保护作用；经热灭活诱导的对虾二次感染WSSV后，28℃环境下病毒增殖速度最快，高温(32℃)和低温(15℃)都会不同程度抑制WSSV的增殖速度。分别采用γ射线灭活和BEI灭活病毒感染对虾并进行二次感染，发现二次感染后，两种方式诱导免疫致敏的实验组比空白对照组的平均存活时间都有了不同程度的提高且差异显著（P＜0.05）。进一步的，对中国对虾免疫致敏相关基因（Dscam）进行了克隆及其在免疫致敏诱导过程中的表达分析。通过比较不同灭活方式诱导对虾产生免疫致敏过程中Dscam基因的表达情况，提示 Dscam基因可能参与了免疫致敏相关的免疫防御体系。使用相同剂量的病毒饲喂或注射感染WSSV后，“黄海2号”比野生对虾的死亡趋势更加缓慢，感染后72 h的半致死剂量更大，说明连续选育显著提高了对虾的抗病能力。通过本项目研究，为首次阐释“类免疫”保护在对虾产生WSSV抗性中的作用奠定了前期理论基础。