Rac蛋白在大黄鱼细胞吞噬中的作用机制

Large yellow croaker (Larimichthys crocea) is one of the most important and largest yield mari-culture fish species in China. However, in recent years, infectious diseases caused by various pathogens are more and more severe. There is an urgent need to understand the mechanism of innate immune in large yellow croaker to control the diseases and for long-term sustainability. Rac protein has been discovered to play an important role in the immune of large yellow croaker by us for the first time, but its role, the pathway and the mechanism in the innate immune is poorly understood. So this project aims at elucidating the mechanism of Rac protein involved in phagocytosis in large yellow croaker，including the following content：1）detecting the changes of phagocytic activity of cells by inhibiting the expression of Rac by RNA interference, overexpression and gene rescue in vitro and in vivo to analyze the role of Rac in phagocytosis; 2）studying the interacted proteins with Rac protein by proteomics techniques including fusion protein GST-pull down, Co-immunoprecipitation (Co-IP), mass spectrometry or amino acid partial sequencing, as well as to identify protein-protein interactions (PPI) by cell co-transfection and Co-IP techniques to explore the pathway of Rac phagocytosis；3) analysis the mechanism of Rac and related proteins in phagocytosis by RNAi and Co-IP techniques, and then predicting three-dimensional structures, interaction models and active sites for PPI of Rac and related proteins. Thus, the function and molecular mechanisms of Rac in large yellow croaker phagocytosis pathway will be revealed, which would enrich the knowledge of Rac in the pahtway of phagocytosis as well as fish immunology, and finally provide a theroretical basis for the molecular breeding of disease resistance and control the diseases in large yellow croaker...

大黄鱼是我国育苗养殖量最大的海水经济鱼类，但病害严重、病因复杂，亟需研究其天然免疫调控机制。申请者率先发现Rac蛋白参与了大黄鱼天然免疫过程，但对其在天然免疫中发挥什么作用、通过什么途径和作用的机理还缺乏了解。因此，本项目拟:1)采用RNAi、过表达和基因拯救等方法，研究Rac 蛋白细胞吞噬活性及抗感染力的关系,分析Rac在细胞吞噬中的作用;2)采用融合蛋白GST-pull down、质谱、免疫共沉淀等蛋白质互作的研究方法，筛选并验证Rac互作蛋白、分析其分子特征,探索Rac在细胞吞噬中的作用通路；3)通过RNAi等技术分析Rac蛋白复合体在大黄鱼细胞吞噬中的作用机理,并利用生物信息学和结构分析方法建构Rac及其互作蛋白的三维结构、蛋白结合活性位点和互作模型。从而揭示Rac蛋白在大黄鱼细胞吞噬的作用机制,本研究可丰富鱼类免疫学知识，为大黄鱼免疫防治和抗病分子育种提供参考依据。

本项目针对申请书提出的3个问题（Rac在大黄鱼免疫中发挥什么作用？作用途径和作用机理是什么？）展开研究，对大黄鱼Rac基因进行克隆与表达、组织表达谱分析、免疫攻毒、攻毒后组织表达变化分析、细胞定位、免疫电镜亚细胞定位、RNA干扰、细胞吞噬活性检测、GST-pull down蛋白质相互作用、质谱分析、互做因子特征与分析等进行了一系列研究，较深入地研究Rac蛋白在大黄鱼细胞吞噬免疫中的作用和相互作用的蛋白质网络和信号通路，揭示了Rac蛋白调控大黄鱼天然免疫的分子机制，丰富了鱼类免疫学知识，为大黄鱼和其它水产动物的免疫防治和抗病分子育种提供新的思路和途径。主要研究成果如下：.1. 采用SMART-RACE技术结合电子克隆获得了大黄鱼Rac基因3个剪接体。进一步研究了组织表达谱特征和免疫刺激后细胞内Rac表达量变化与细胞吞噬活性及抗感染水平之间的关系，表明Rac在大黄鱼细胞吞噬中发挥作用。.2. 采用融合蛋白GST-pull down、质谱/氨基酸部分测序等技术，结合生物信息学分析筛选细胞吞噬途径中Rac的互作蛋白有原肌球蛋白（Tropomyosin，TPM）、QM、Rab、酪氨酸激酶（LcSyk）。.3. 研究了互做因子原肌球蛋白（Tropomyosin，TPM）、QM、Rab、酪氨酸激酶（LcSyk）的基因序列、时空表达谱、分子特征和免疫特征，并进行了蛋白质的表达及定位。.4. 通过蛋白质相互作用、RNA干扰技术和细胞吞噬等实验研究了Rac蛋白复合体在大黄鱼细胞吞噬中的作用。基于Rac蛋白在免疫刺激后表达量明显上调，其互做蛋白原肌球蛋白（Tropomyosin，TPM）、QM、Rab、酪氨酸激酶（LcSyk）都是天然免疫中吞噬作用的关键蛋白，提出Rac在大黄鱼细胞吞噬信号通路中发挥关键作用。.5. 发表论文7篇，授权1篇国家发明专利，培养研究生3名（2名已经顺利毕业，1名2018年毕业）。.一些实验结果目前正在整理补充中，后续会以论文的形式发表提交。