牙鲆JNK2基因的表达分析及免疫功能探究

Expression Analysis and Functional Characterization of JNK2 in Japanese Flounder(Paralichthys olivaceus)

本研究利用实验室已建牙鲆(Paralichthysolivaceus)转录组数据库,得到牙鲆JNK2基因(Po JNK2)并利用PCR技术进行序列验证。PoJNK2的开放阅读框长度为1263 bp,编码420个氨基酸;通过SMART服务器对PoJNK2的结构域进行预测,显示PoJNK2具有典型的丝氨酸-苏氨酸蛋白激酶催化结构域S_TKc。利用q RT-PCR分析PoJNK2在牙鲆成体不同组织中的表达水平,发现其在牙鲆免疫组织中均有表达,因此,初步推测PoJNK2在牙鲆免疫应答方面可能发挥作用。本研究设计了体内迟缓爱德华氏菌(Edwardsiellatarda)侵染实验和体外免疫刺激细胞实验以探究PoJNK2在牙鲆免疫反应中的作用。在迟缓爱德华氏菌体内注射牙鲆成体后,PoJNK2在脾脏、头肾和鳃中有不同程度的表达上调。同时,使用迟缓爱德华氏菌、Poly I:C和肿瘤坏死因子-α(TNF-α)刺激牙鲆鳃细胞系后,发现PoJNK2在不同时间点表达上调。除此之外,通过细胞转染实验在牙鲆鳃细胞系中过表达PoJNK2,发现其对下游炎症细胞因子的表达及激活蛋白Activator protein-1 (AP-1)的转录活性具有显著的上调作用,进一步阐明了PoJNK2在调节牙鲆免疫应答方面的作用。

The immune system of teleost,similar to mammals,is divided into two subsystems:Innate immune system and acquired immune system.Acting as the first line of defense against invading pathogens and pathogenicity factors,the innate immune system can be activated by the pattern recognition receptor(PRRs)which can recognize the pathogen-associated molecular pattern(PAMPs)and activate the downstream inflammatory response accordingly.MAPK(mitogen-activated protein kinase)signaling pathway is an important signal transduction system in eukaryote cells,which can mediate extracellular signaling into the cell and participate in various cellular processes such as cell proliferation,apoptosis,and immune response.JNK(c-Jun N-terminal kinase)signal pathway is an important branch of MAPK signal pathway.JNK have been extensively studied in mammals,but not adequately in the teleost.In this research,we designed experiments to study the role of JNK2 genes in the antimicrobial reaction of Japanese flounder and their effects on the downstream of the pathway in order to provide a theoretical basis for the study of JNK signaling pathway in fish.In this study,the JNK2 gene sequences of Japanese flounder were obtained from the transcription database of flounder and named after PoJNK2.The PoJNK2 ORF region encodes is 1263 bp and encodes 420 amino acids.The domains of PoJNK2 genes were predicted by SMART server.The results showed that the JNK2 gene had a typical serine-threonine protein kinase domain S_TKc.The expression levels of PoJNK2 genes in different tissues of flounder were analyzed by the qRT-PCR technique.It was found that PoJNK2 were widely expressed in the immune tissue of Japanese flounder.Therefore,it was preliminarily speculated that PoJNK2 played a major role in the immune response of Japanese flounder.We designed an infection experiment of Edwardsiella tarda in vivo and immune stimulatory cell experiments in vitro to investigate the role of PoJNK2 in the immune response of Japanese flounder.The expression of PoJNK2 was up-regulated in the spleen,kidney,and gill of Japanese flounder after injecting E.tarda in vivo.At the same time,the gill cells of flounder(FG)were stimulated by E.tarda,Poly I:C and TNF-α.The expression of PoJNK2 was up-regulated at different time points.In addition,we overexpressed PoJNK2 in FG by cell transfection and it was found that PoJNK2 had significant regulatory effects on the regulation of downstream pro-inflammatory cytokines and transcription factor AP-1.The role of PoJNK2 in regulating the immune response of Japanese flounder was further clarified.