长牡蛎干扰素调节因子免疫功能及作用机制研究

Viral diseases are serious threats to shellfish aquaculture. And the intensive study of host immune defense mechanism is needed for prevention and control of diseases. Interferon regulatory factors (IRFs) are a family of key transcription factors which could participate in RLR or TLR antiviral signaling pathway. However, seldom researches were carried out in order to decipher immune function of IRFs in invertebrates. In the present project, we plan to reveal the antiviral function and related mechanism of IRFs in the Pacific oyster Crassostrea gigas. On the basis of verifying the antiviral immune function of oyster IRFs, the activation mechanism, subcellular localization and dimerization, and their downstream target genes of IRFs would be researched. Firstly, the proteins that interact with oyster IRFs will be validated in order to determine their activation of oyster IRFs. Secondly, the characteristics, like subcellular localization and dimerization, of IRFs after activation will be studied to identify the molecular basis of IRFs for antiviral immune function. Finally, the downstream target genes of IRFs will be screened and verified by chromatin immunoprecipitation sequencing and some other technologies, in order to explore the gene regulation network of oyster IRFs. The research results will not only be helpful for revealing the immune function of invertebrates IRFs and enriching the invertebrate innate immune theory, but also provide theoretical basis for virus-resistance molecular breeding of oyster.

病毒性疾病是贝类养殖产业的严重威胁，病害防控需对宿主免疫机制进行深入解析。干扰素调节因子IRF是一类参与RLR、TLR等抗病毒天然免疫通路的关键转录因子。目前关于贝类IRF免疫功能及机制的研究还很匮乏。本项目以长牡蛎为研究对象，开展IRF免疫功能及作用机制的研究。在证实长牡蛎IRF抗病毒免疫功能的基础上，对IRF的激活机制、激活后入核与二聚化调控机制以及下游靶基因等方面进行研究。首先，鉴定长牡蛎IRF互作蛋白，验证互作蛋白对IRF的激活作用；其次，研究长牡蛎IRF激活后入核与二聚化调控特征，进一步解析IRF抗病毒天然免疫的分子机制；最后，通过染色质免疫共沉淀结合测序等技术筛选并验证长牡蛎IRF蛋白调控的下游靶基因，探索长牡蛎IRF基因调控网络。该项目的顺利实施将有助于解析无脊椎动物IRF免疫功能及作用机制，丰富无脊椎动物天然免疫理论，同时也为牡蛎的病害防治及抗病品系的选育提供理论支持。

近年来，病毒性疾病对贝类养殖产业造成严重威胁，而对贝类免疫机制的解析是病害防控的基础和关键。干扰素调节因子IRF及核因子-κB（NF-κB）等是参与RLR、TLR等抗病毒天然免疫通路的关键转录因子。目前关于贝类IRF、NF-κB免疫功能及调控机制的研究还很匮乏。本项目以长牡蛎为研究对象，聚焦RLR、TLR等信号通路信号转导及IRF、NF-κB激活调控等作用机制的研究。着重研究了IRF、NF-κB关键调控蛋白IKK家族在介导免疫信号传递及IRF、NF-κB激活方面功能。（1）长牡蛎经典IKK家族基因-IKKα/β，其编码蛋白参与TLR、RLR通路信号转导并与长牡蛎IRF蛋白发生互作，进而激活ISRE、NF-κB报告基因。（2）长牡蛎IKK相关激酶家族基因-IKKε同样为关键的信号转导分子，且与长牡蛎IRF8蛋白直接互作，可激活ISRE、NF-κB报告基因。另一IKK相关激酶基因TBK1/IKKε也参与TLR、RLR信号转导，只是在IRF、NF-κB激活过程中可能发挥了负调控的作用。（3）鉴定了新的NF-κB家族基因（CgRel2）及其调控基因IκB（CgIκB4）。研究发现表明CgRel2可能作为关键转录因子参与宿主天然免疫，而CgIκB4可能通过调节NF-κB家族蛋白的活性而在长牡蛎天然免疫中发挥重要作用。本项目研究结果对于解析无脊椎动物RLR、TLR天然免疫通路的信号转导机制及IRF、NF-κB激活及调控机制，丰富无脊椎动物天然免疫理论有巨大贡献，同时也为牡蛎的病害防治及抗病品系的选育等提供理论支持。