内含子保留形成的Relish可变剪接异构体在克氏原螯虾先天免疫中的功能研究

Relish transcription factor plays an essential role in innate immunity. Relish gene in insects can generate multiple alternatively spliced isoforms and perform different functions, but that is rarely reported in crustaceans. We have identified a short alternatively spliced isoform of Relish with retained intron (SPcRelish) and a long isoform of Relish (LPcRelish) in Procambarus clarkii. Based on this, this project intends to: 1) Knock down of these two genes by RNA interference (RNAi) in vivo will be conducted to confirm the roles (positive or negative) of them in innate immunity; 2) If SPcRelish plays a positive regulatory role, we will clarify the relationship between SPcRelish and intestinal microbial stability by RNAi and metagenomic sequencing. 3) If SPcRelish exerts a negative regulatory role, interaction between SPcRelish and LPcRelish will be studied by pull-down, Far-western and coIP. Meanwhile, over-expression in Drosophila S2 cells will be utilized to explore their effects on the activation of antimicrobial peptide promoters. We will for the first time clarify the function of alternatively spliced isoform of Relish with intron retention in innate immunity. This project will expand the knowledge of innate immune responses and provide fresh ideas for the development of effective prevention strategies for crustacean disease.

Relish转录因子在先天免疫中发挥重要作用。昆虫Relish存在多种可变剪接异构体并发挥不同的功能，但在甲壳动物中却鲜有报道。申请人在克氏原螯虾体内鉴定到一个内含子保留形成的短型Relish可变剪接异构体（SPcRelish）和一个长型Relish异构体（LPcRelish）。基于此，本项目拟：1）体内干扰这两个基因，明确它们在先天免疫中的作用（正向或负向）；2）若SPcRelish发挥正调控作用，则利用干扰手段和宏基因组测序，理清其与肠道菌群稳定性之间的关系；3）若SPcRelish发挥负调控作用，则通过Pull-down、Far-western和coIP研究SPcRelish与LPcRelish的相互作用，并利用过表达探清它们对抗菌肽启动子激活的影响。本项目将首次阐明内含子保留形成的Relish可变剪接异构体在先天免疫中的功能，拓展先天免疫知识体系，并为甲壳动物病害防治提供新的思路。

本项目是在前期发现克氏原螯虾体内存在一个组成型Relish可变剪接异构体（LPcRelish）和一个内含子保留型Relish异构体（SPcRelish）的基础上申请的，拟对LPcRelish和SPcRelish进行鉴定和时空表达分析，研究它们在先天免疫调控中的作用（正向或负向），并探索SPcRelish发挥正向或负向调控作用的机制。立项后，我们对LPcRelish和SPcRelish的分子特征、组织分布、表达模式以及在先天免疫调控中的作用进行了系统研究。结果发现，LPcRelish和SPcRelish是通过选择性剪接产生的。LPcRelish的编码区由1–25个外显子组成，而SPcRelish cDNA由外显子1–9和部分内含子9组成。LPcRelish蛋白包含RHD、IPT、低复杂度区域、卷曲螺旋和ANK，而SPcRelish蛋白仅包含RHD和IPT结构域。副溶血弧菌感染能够激活两个Relish异构体的表达。并且，在弧菌感染过程中，LPcRelish和SPcRelish对多种抗菌肽的表达存在不同的调控作用。其中，LPcRelish对多种抗菌肽的表达均呈现正调控作用，而SPcRelish对不同抗菌肽的表达呈现正调控、负调控或者无影响的多样调节功能，表明在克氏原螯虾体内存在复杂的抗菌应答网络。本研究首次对内含子保留形成的Relish可变剪接异构体进行了分子鉴定和功能研究，阐明了其在先天免疫防御中的作用，拓展了先天免疫知识体系，并为甲壳动物病害防治提供了新的思路。此外，在本项目支持下，我们还发现1）克氏原螯虾C型凝集素和Relish之间存在正反馈回路，促进抗菌肽的表达；2）克氏原螯虾中存在一条响应WSSV感染的FOXO-Relish-Cathepsin L调控途径。另外，还阐释了日本沼虾Relish的表达调控机制及功能。本项目成果以项目负责人通讯作者身份发表SCI论文6篇。