细胞膜NF-κB（mNF-κB）信号通路及其介导的凡纳滨对虾抗真菌免疫调控机制

The NF-κB family is a group of important transcription factors, involving in regulation of most biological processes in animals. The Pacific White Shrimp, Litopenaeus vannamei, is a representative species of the Class Crustacea, and is also one of the major species of the global shrimp aquaculture industry. The fungus is the important pathogen that threatens the shrimp aquaculture, but the study on the regulatory mechanism of antifungal immunity in shrimp is almost blank at present. We have found a novel member of the NF-κB family from L. vannamei, termed membrane NF-κB (mNF-κB), that is located on the cell membrane. The preliminary study has showed that mNF-κB could be involved in regulation of antifungal immunity in shrimp. In this project, we plan to study the protein nature, evolutionary status and transcription factor function of mNF-κB, and further reveal the basic framework and regulatory mechanism of the mNF-κB signaling pathway through screening and identifying the target genes and upstream regulatory molecules of mNF-κB. Moreover, we plan to analyze the crosstalk between the mNF-κB pathway and other immune signaling pathways, and further study the mechanism mediated by the mNF-κB pathway to regulate the shrimp antifungal immunity. This project helps to fill the research gap in the field of shrimp antifungal immunity, and makes a theoretical foundation for preventing fungal diseases in aquaculture shrimp, and can provide an important supplement to the research on the field of NF-κB pathways in organisms.

NF-κB家族是一类重要的转录因子，参与了动物体内大多数生命进程的调控。凡纳滨对虾是甲壳纲中具有代表性的物种，也是全球主要的水产养殖虾类之一。真菌是威胁对虾养殖的重要病原，但目前对对虾抗真菌免疫调控机制的研究几乎空白。我们在凡纳滨对虾中发现了一种定位于细胞膜上的NF-κB家族新成员，命名为细胞膜NF-κB（membrane NF-κB, mNF-κB）。初步研究发现mNF-κB可能参与了对虾抗真菌免疫调控。本项目拟对mNF-κB的蛋白特征、进化地位及转录因子功能进行研究，通过筛选及鉴定其靶基因和上游调控分子，揭示mNF-κB信号通路的基本框架及调控机理；进一步解析该通路与其他免疫信号通路间的交互作用，并对其介导的对虾抗真菌免疫调控机制进行研究。本项目有助于填补目前在对虾抗真菌免疫领域的研究空白，为养殖对虾真菌病害的防治提供理论基础，并能够为生物界中NF-κB通路领域的研究提供重要的补充。

mNF-κB是在凡纳滨对虾中发现的具有C末端跨膜区的核因子κB（NF-κB）家族蛋白新成员。本项目针对mNF-κB的蛋白特征、进化地位、转录因子功能、参与的信号通路及其免疫调控作用进行研究，取得以下成果：发现mNF-κB蛋白主要定位于细胞膜和线粒体外膜上，且表达于对虾所有的组织中；通过同源序列分析、进化树构建以及DNA结合位点的鉴定，证明mNF-κB是NF-κB家族中一支独立进化的新成员；mNF-κB主要响应革兰氏阳性菌及其病原相关分子模式（PAMP）脂磷壁酸（LTA）的刺激而激活，其活化标志是全蛋白经Caspase Dronc和Caspase A的剪切而释放N端区域入核；胞外蛋白Spätzle 2/4、细胞膜受体Toll3、及胞内分子MyD88、TRAF3、TRAF6、TAK1、IKK-β和IKK-ε构成了mNF-κB信号通路的基本框架；通过miRNA-10c、FoxP、LARK等分子的介导，mNF-κB通路与其他信号通路之间发生交互作用；我们进一步鉴定了mNF-κB信号通路的两个调控靶基因抗菌肽PEN4和Crustin1，并证明该通路在对虾抗真菌、抗病毒和抗细菌免疫中均发挥重要作用。本项目在研期间，共发表论文16篇，提交专利申请2项，项目参与者7人次参加了国内外学术会议，培养毕业博士研究生2人、毕业硕士研究生3人。