牙鲆GATA3对免疫应答的调控作用

Teleost, as an inferior vertebrate, is a key node in evolution. It is a good model to study the functions of immune response components based on the earliest vertebrates possessing both innate and adaptive immunity. Japanese flounder (Paralichthys olivaceus) is an important cultural species, but diseases break out frequently during cultivation. The increasing usage of antibiotic causes water pollution and resistance of pathogens, which causes inconsistency with the healthy cultivation concept. This study aims to explore the regulatory effects of GATA3 on lymphocytes in Japanese flounder, and to reveal the immune response related to GATA3 to pathogens. Firstly, the expression pattern of GATA3 in immune organs and lymphocytes is about to be detected by qRT-PCR and western blotting. Secondly, the changes of GATA3 to pathogenic challenge are detected in vivo and in vitro. Thirdly, overexpression experiment and knockdown experiment will be performed to explore the effect of GATA3 on its downstream genes. Moreover, ChIP-seq will be used to select target genes of GATA3 in different types of lymphocytes. Then, the interaction of GATA3 and its target genes will be verified. The mutation plasmid will be constructed to investigate the functional domain of GATA3. Our study will help to improve the knowledge on the immune system regarding GATA3 and provide a theoretical basis for the study of teleost immunity.

鱼类作为低等的脊椎动物，在进化方面处于节点位置，因其是最早分化出先天免疫和获得性免疫的脊椎动物，是研究免疫的良好模型。牙鲆作为重要的经济物种，在工厂化养殖过程中病害频发，伴随抗生素等药物的滥用，造成水体污染和病原微生物产生抗药性等不良后果，与生态健康养殖理念不符。本研究旨在通过探究GATA3在牙鲆中对淋巴细胞的调控作用从而发挥免疫功能，解析GATA3对免疫应答的调控作用。首先检测GATA3在免疫器官和淋巴细胞中的分布；其次通过体外和体内实验检测GATA3对病原微生物刺激的免疫反应；再次通过GATA3过表达和RNAi敲降检测对下游基因的影响；最后通过ChIP-seq对GATA3的靶基因进行筛选，并对GATA3和靶基因的互作进行验证，同时构建GATA3结构域缺失突变质粒，明确哪些结构域在调控过程中发挥功能，阐明GATA3是如何与靶基因互作发挥其功能，为鱼类免疫研究提供一定的理论基础。

鱼类作为低等的脊椎动物，在进化方面处于节点位置，因其是最早分化出先天免疫和获得性免疫的脊椎动物，是研究免疫的良好模型。牙鲆作为重要的经济物种，在工厂化养殖过程中病害频发，伴随抗生素等药物的滥用，造成水体污染和病原微生物产生抗药性等不良后果，与生态健康养殖理念不符。在本研究中获得了牙鲆GATA3基因的序列和在胚胎发育时期、成鱼组织中的表达模式，发现该基因在免疫相关组织中呈现高表达趋势，同时发现受到病原微生物刺激后，在体内和体外实验中都会发现GATA3产生免疫应答，产生免疫抑制现象；另外通过免疫组化实验，定位了GATA3蛋白在鳃和头肾中的表达位置；对GATA3的靶基因进行了筛选和验证，获得了200多个靶基因，经验证，GATA3和这些靶基因的启动子区存在直接互作关系；过表达实验发现，GATA3可调控下游基因的表达。这些发现为鱼类免疫研究提供一定的理论基础。