泥蚶JNK基因在哈维氏弧菌感染下的表达特征及其对激活蛋白-1的调控分析  

Expression characteristics of the JNK gene in Tegillarca granosa infected by Vibrio harveyi and its regulation on activating protein-1

作　　者：王秋金 肖国强[1,2] 滕爽爽 WANG Qiujin;XIAO Guoqiang;TENG Shuangshuang(Zhejiang Mariculture Research Institute,Zhejiang Key Laboratory of Exploitation and Preservation of Coastal Bio-Resource,Wenzhou Key Laboratory of Marine Biological Genetics and Breeding,Wenzhou 325005,China;State-level Fisheries Science Experimental Teaching Demonstration Center,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]浙江省海洋水产养殖研究所,浙江省近岸水域生物资源开发与保护重点实验室,温州市海洋生物遗传育种重点实验室,浙江温州325005 [2]上海海洋大学,水产科学国家级实验教学示范中心,上海201306

出　　处：《中国水产科学》2023年第1期60-74,共15页Journal of Fishery Sciences of China

基　　金：浙江省自然科学基金项目(LY23C190006);台州市重大农业科技“揭榜挂帅”研发攻关计划项目(NYJBGS202203);温州市农业新品种选育协作组项目(2019ZX001-1);现代农业产业技术体系建设专项(CARS-49).

摘　　要：泥蚶(Tegillarca granosa)作为我国常见的滩涂养殖贝类,具有较高的经济价值。以弧菌为代表的条件致病菌一直以来是困扰双壳贝类健康养殖的一大难题。为了探讨泥蚶感染弧菌过程中,c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)介导的激活蛋白-1(activating protein-1,AP-1)先天免疫防御通路的作用及机制,本研究采用PCR技术克隆了泥蚶JNK基因(TgJNK)的c DNA序列,并对TgJNK和TgAP-1在弧菌感染下的表达和调控关系进行了分析。结果显示,TgJNK基因c DNA全长2696 bp,开放阅读框1332 bp,编码了443个氨基酸;蛋白多重比较和邻近系统发育树分析表明,TgJNK相对保守,存在一个STKc-JNK结构域,与长牡蛎(Crassostrea gigas)和紫贻贝(Mytilus edulis)的同源相似度较高。q RT-PCR和WB结果表明TgJNK在泥蚶的鳃中表达最高。哈维氏弧菌(Vibrio harveyi)的浸泡感染可显著上调TgJNK的转录水平(P<0.05)和磷酸化水平,并增加TgAP-1的蛋白丰度;JNK抑制后TgJNK和TgAP-1的转录水平显著降低(P<0.05),TgJNK磷酸化水平和TgAP-1蛋白丰度也降低。CoIP分析显示,磷酸化的JNK蛋白能与TgAP-1蛋白相互结合。研究表明,TgJNK和TgAP-1共同参与了泥蚶感染弧菌后的先天免疫反应,TgJNK蛋白磷酸化是激活TgAP-1蛋白的重要因素,TgJNK能够正向调控TgAP-1的表达。本研究阐明了泥蚶先天免疫通路JNK介导的AP-1途径在弧菌感染过程中的信号传递机制,丰富了泥蚶等双壳贝类先天免疫理论知识。Blood clam(Tegillarca granosa),as a common farmed bivalve in tidal flat shellfish,has high economic value.Conditional pathogenic bacteria,such as Vibrio,is a serious problem encountered in the healthy culture of bivalve shellfish.To explore the role and mechanism of JNK-mediated AP-1 innate immune defense pathway in the process of vibrio infection,the cDNA sequence of TgJNK was cloned by PCR,and the expression and regulatory relationship of TgJNK and TgAP-1 after vibrio infection were analyzed.The results showed that the fulllength of the TgJNK cDNA sequence was 2696 bp with an open reading frame of 1,332 bp,encoding a 443 amino acid polypeptide.Multiple protein comparison and adjacent phylogenetic tree analysis showed that TgJNK was relatively conservative with a STKc-JNK domain and had high homologous similarity with Crassostrea gigas and Mytilus edulis.The qRT-PCR and WB analysis showed that TgJNK was highly expressed in the gill.The level of TgJNK transcription was up-regulated significantly(P<0.05)with immersion infection of Vibrio harveyi.At the same time,the phosphorylation level of TgJNK and the abundance of TgAP-1 protein increased under the stimulation of vibrio.The effect of the JNK inhibitor could significantly decrease the transcription level of TgJNK and TgAP-1(P<0.05).Correspondingly,the phosphorylation of TgJNK and the abundance of TgAP-1 protein decreased after JNK inhibition.In addition,CoIP analysis showed that the phosphorylated JNK protein could bind to TgAP-1 protein.These results indicated that TgJNK and TgAP-1 participated in the innate immune response of Vibrio infection in blood clams.The phosphorylation of TgJNK protein was an important factor in activating TgAP-1 protein,and TgJNK could positively regulate the expression of TgAP-1.The study elucidated the signal transduction mechanism of JNK-mediated AP-1 pathway after vibrio infection and enriched the theoretical knowledge on the innate immunity of bivalve shellfish.

关 键 词：泥蚶 JNK AP-1 弧菌感染 磷酸化调控 

分 类 号：S941[农业科学—水产养殖]