摘要
为探究转化生长因子β激活激酶1基因(TAK1)在硬骨鱼先天免疫中的重要作用,本研究基于实验室已建的牙鲆(Paralichthys olivaceus)转录组数据库,对牙鲆TAK1基因(PoTAK1)进行了克隆及验证。研究显示,其开放阅读框(ORF)长为1 728 bp,编码575个氨基酸,蛋白分子量(Mw)约为64.33 kDa,理论等电点(pI)为6.66,编码的蛋白在N端具有一个S_TKc结构域,在C端具有一个卷曲螺旋区域(Coiled coil region)。氨基酸多序列比对和系统发育分析表明,TAK1基因在脊椎动物中高度保守。组织表达分析显示,PoTAK1在肝脏和鳃中的表达量较高;体内攻毒实验表明,被迟缓爱德华氏菌(Edwardsiella tarda)感染后,PoTAK1在脾脏和头肾中的表达量均显著上调;体外免疫刺激实验表明,Poly I:C、TNF-α和迟缓爱德华氏菌刺激后,在各时间点牙鲆鳃细胞系中PoTAK1的表达量均显著上调。亚细胞定位显示,PoTAK1主要在细胞质中表达。双萤光素酶报告实验证明,PoTAK1能够激活MAPK通路下游AP-1转录因子的活性。体外免疫调节检测实验表明,过表达PoTAK1能够上调AP-1下游IL-1β、IL-6、IL-8和TNF-α等促炎细胞因子的转录表达。研究结果表明,PoTAK1参与了牙鲆的先天免疫反应,并对其具有一定的调节作用。本研究有助于更好地理解PoTAK1在牙鲆抵抗外界病原体入侵过程中的作用机制。
To investigate the important role of transforming growth factor β-activated kinase 1 gene(TAK1) in innate immunity of teleost, the Japanese flounder TAK1 gene(PoTAK1) was cloned and verified based on the established transcriptome database of Japanese flounder in this study. The results showed that the length of open reading frame(ORF) of PoTAK1 was 1 728 bp, which encoded 575 amino acids. The protein molecular weight(Mw) of PoTAK1 was approximately 64.33 kDa, and the theoretical isoelectric point(pI) of PoTAK1 was 6.66. The encoded protein of PoTAK1 possesses a S_TKc domain at the N-terminal and a coiled-coil region at the C-terminal. Amino acid multiple sequence alignment and phylogenetic analysis showed that TAK1 gene was highly conserved in vertebrates. Tissue expression analysis exhibited that the expression level of PoTAK1 was relatively high in liver and gill. The challenge experiment in vivo showed that the expression of PoTAK1 in spleen and head kidney was significantly up-regulated after Edwardsiella tarda infection. The immunostimulation experiment in vitro showed the expression of PoTAK1 was significantly up-regulated in Japanese flounder gill cell line at each time point after Poly I:C, TNF-α and E. tarda stimulation. Subcellular localization showed that PoTAK1 was mainly expressed in cytoplasm. Dual luciferase assay demonstrated that PoTAK1 can activate the activity of AP-1 transcription factor downstream of MAPK pathway. Immunomodulation assay in vitro demonstrated that the overexpression of PoTAK1 was able to up-regulate the transcriptional expression of pro-inflammatory cytokines downstream of AP-1, such as IL-1β, IL-6, IL-8, and TNF-α. The above findings indicated that PoTAK1 was involved in the innate immune response of Japanese flounder and possessed a certain regulatory role on it. In conclusion, our findings should aid to better understand the mechanism of PoTAK1 in the process of Japanese flounder against to the invasion of external pathogens.
作者
李恒顺
司瑜
王宣刚
曲江波
王志刚
刘金相
张全启
于海洋
Li Hengshun;Si Yu;Wang Xuangang;Qu Jiangbo;Wang Zhigang;Liu Jinxiang;Zhang Quanqi;Yu Haiyang(Key Laboratory of Marine Genetics and Breeding,Ministry of Education,Ocean University of China,Qingdao 266003,China;Key Laboratory of Tropical Aquatic Germplasm of Hainan Province,Sanya Oceanographic Institution,Ocean University of China,Sanya 572000,China;Laboratory for Marine Fisheries Science and Food Production Process,Pilot National Laboratory for Marine Science and Technology(Qingdao),Qingdao 266237,China)
出处
《中国海洋大学学报（自然科学版）》
CAS
CSCD
北大核心
2023年第3期152-165,共14页
Periodical of Ocean University of China
基金
国家自然科学基金项目(31802327)资助。
关键词
牙鲆
转化生长因子β激活激酶1
先天免疫
迟缓爱德华氏菌
基因表达
Paralichthys olivaceus
transforming growth factorβ-activated kinase 1
innate immunity
Edwardsiella tarda
gene expression
