拟穴青蟹特异性免疫致敏的分子机制及眼柄在其中的作用

White Spot Syndrome Virus (WSSV) and Vibrio parahaemolyticus cause crab disease frequently. Traditionally, it is believed that as invertebrates, crabs do not possess specific immunity, thus it is impossible to develop immune prevention for crabs. Recently, a phenomena named specific immune priming was discovered in Drosophila and other invertebrates, but the molecular mechanism is unclear. In our previous project we surprisingly found a large number of immune gene in the eyestalk, an important component of a hypothalamic - pituitary - gonadal (HPG) like axis that plays an important role in the regulation of reproduction in the crab, which suggests that the crab eyestalk not only acts as HPG to regulate reproductive generally, but may also have immune function. In vertebrates, the regulatory role of miRNA in specific immunity has been well recognized, but the role of miRNA in the specific immune priming in invertebrates has never been studied. This project intends to use the next-generation sequencing technique combined with bioinformatics analysis to identify deferentially expressed transcripts (including protein-coding transcripts and miRNAs) between: 1) immunized and non-immunized groups, 2) primary and secondary immunized groups, 3) WSSV and V. parahaemolyticus immunized groups, and 4) eyestalk-intact and eyestalk-ablated immunized groups, then further confirm these results by real-time quantitative PCR, in situ hybridization, functional analysis, etc. so that we will be able to understand the molecular mechanisms of specific immune priming in crab and the role of eyestalks therein. Completion of this project will lay the foundation for the establishment of immune prevention for crab.

白斑病毒（WSSV）和副溶血弧菌常引起养殖青蟹的病害。传统观点认为作为无脊椎动物的蟹类没有特异性免疫，无法建立免疫防治。新近在无脊椎动物中发现了特异性的免疫致敏，但分子机制不清。在前期工作中我们意外地发现作为类似于下丘脑-垂体-生殖腺轴（HPG）的一部分，在蟹的生殖调控中起作用的青蟹眼柄中有大量的免疫基因表达，提示青蟹的眼柄除了可调控生殖外，还具有免疫功能。miRNA在脊椎动物的特异性免疫中起调节作用，但在无脊椎动物特异性免疫致敏中的作用未曾研究。本项目拟利用二代测序结合生物信息学分析筛选青蟹在1）免疫组与非免疫组，2）初次免疫组与再次免疫组，3）WSSV免疫组和副溶血弧菌免疫组，4）眼柄摘除免疫组与未摘除免疫组的差异表达转录本（含蛋白和miRNA），并用实时定量PCR、原位杂交等加以确认，可以使我们在分子水平了解青蟹免疫致敏的机制及眼眼柄在其中的作用。为建立青蟹疾病的免疫防治奠定基础。

拟穴青蟹是我国最重要的海水养殖蟹类之一，病原生物感染引起的病害给青蟹养殖业造成了严重损失。传统观点认为作为无脊椎动物的蟹类没有特异性免疫，无法建立免疫防治。新近在无脊椎动物中发现了特异性的免疫致敏，但分子机制不清。青蟹是否具有特异性的免疫致敏机制是一个重要的学术问题。本项目通过实验统计了初次和二次免疫过程中拟穴青蟹存活率变化，测定了ALP、ACP等在免疫致敏过程中的变化，初步证实了青蟹免疫致敏机制的存在。构建了拟穴青蟹全长转录组，分析了多个免疫致敏的转录组，获得了多个差异表达转录子。分析、发掘了一些免疫致敏相关基因，如Toll家族基因、溶菌酶基因，免疫相关信号通路中的基因，抗菌肽，酚氧化酶原激活系统相关基因以及靶基因为免疫致敏相关基因的miRNA等。对甲壳动物中参与病原识别的Toll样受体家族的研究进行了回顾总结，在全基因组水平鉴定了凡纳滨对虾的全部Toll样受体家族成员及它们对副溶血弧菌感染的反应，鉴定、分析了一个拟穴青蟹新型抗菌肽。克隆分析了可能受免疫致敏机制调控的ALP及其启动子活性。分析了虾眼柄在免疫中的作用。分析了多个拟穴青蟹参与免疫作用的miRNA。提出了可能参与青蟹免疫致敏基因的表达特点及作用网络。通过本项目的研究，使我们初步在分子水平了解青蟹免疫致敏的机制及眼眼柄在其中的作用。可为建立青蟹疾病的免疫防治奠定基础。