三疣梭子蟹C-型凝集素CTL5基因的克隆及在病原刺激下的免疫应答  

Cloning and response to pathogen stimulation of C-type lectin gene CTL5 in swimming crab Portunus trituberculatus

作　　者：阎德平 吕建建 题兴斌[3] 张文 张云滨 宋柳 刘萍 YAN Deping;LU Jianjian;TI Xingbin;ZHANG Wen;ZHANG Yunbin;SONG Liu;LIU Ping(College of Fisheries and Life Science,Dalian Ocean University,Dalian 116023,China;Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao National Laboratory for Marine Science and Technology,Qingdao 266071,China;Key Laboratory of Sustainable Development of Marine Fisheries,Ministry of Agriculture and Rural Affairs,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China)

机构地区：[1]大连海洋大学水产与生命学院,辽宁大连116023 [2]青岛海洋科学与技术国家实验室,海洋渔业科学与食物产出过程功能实验室,山东青岛266071 [3]中国水产科学研究院黄海水产研究所,农业农村部海洋渔业可持续发展重点实验室,山东青岛266071

出　　处：《大连海洋大学学报》2020年第2期253-259,共7页Journal of Dalian Ocean University

基　　金：国家自然科学基金资助项目(41576147);泰山领军人才工程高效生态农业创新类计划项目(LJNY2015002);国家虾蟹产业技术体系专项(CARS-48);江苏省重点研发计划项目(BE2017325);青岛市应用基础研究计划项目(17-1-1-95-jch);中央级公益性科研院所基本科研业务费项目(20603022018027)。

摘　　要：为明确三疣梭子蟹Portunus trituberculatus C-型凝集素CTL5的结构和功能,采用cDNA末端快速扩增(Rapid amplification of cDNA end,RACE)技术克隆获得三疣梭子蟹C-型凝集素CTL5基因(PtCTL5)的cDNA全长。结果表明:PtCTL5基因全长为1026 bp,包含732 bp的开放阅读框,编码了243个氨基酸组成的蛋白,相对分子质量为26000,为半乳糖结合型凝集素;Blast分析显示,PtCTL5氨基酸序列与克氏原螯虾Procambarus clarkia CTL的一致性最高,为41.84%;系统进化树分析显示,PtCTL5与已发表的三疣梭子蟹的另4种C-型凝集素亲缘关系较远;胚胎及幼体不同发育时期的定量分析表明,PtCTL5在溞状幼体Ⅲ期表达量最高,在胚胎发育的心跳期表达量最低;组织表达分析显示,PtCTL5在血细胞中的表达量最高,肝胰腺中次之;人工注射副溶血弧菌Vibrio parahemolyticus后,PtCTL5在血细胞和肝胰腺中的表达量均于72 h时达到峰值;而注射对虾白斑综合征病毒(WSSV)感染后,PtCTL5在血细胞和肝胰腺中的表达量分别于72、12 h达到峰值且显著高于对照组(P<0.05)。研究表明,C-型凝集素在三疣梭子蟹先天免疫中具有一定的功能,本研究结果可为进一步解析三疣梭子蟹免疫应答机制提供参考。The C-type lectin cDNA of swimming crab Portunus trituberculatus(designated PtCTL5)was cloned by rapid amplification of cDNA ends to clarify the structure and function of PtCTL5 gene.It was found that PtCTL5 as galactose-binding lectin was 1026 bp in length,containing an open reading frame of 732 bp and encoding a mature protein of 243 amino acids,with a relative molecular mass of 26000.Blast analysis revealed that amino acid sequence of PtCTL5 had the maximal similarity with red swamp crayfish Procambarus clarkii,up to 41.84%.Phylogenetic tree analysis showed that PtCTL5 was far from being related to four other C-type lectins found from swimming crab Portunus trituberculatus.The expression of PtCTL5 gene was shown to be varied with different developmental stages,the maximum in zoeaⅢstage of larval development and the minimal in heart beating stage of embryo development.The PtCTL5 gene was expressed in all tested tissues with the maximum in hemocytes,followed by hepatopancreas.Under normal circumstances,after artificial infection with Vibrio parahaemolyticus,the maximal expression of PtCTL5 gene in hemocytes and in hepatopancreas at 72 h;while,exposed to WSSV infection,the peak PtCTL5 gene expression was observed hemocytes and hepatopancreas at 72 h and 12 h,respectively,significantly higher than that in the control group(P<0.05).The findings indicate that PtCTL5 plays an important role in the immune response in swimming crab and lays a reference for further research on the swimming crab immune system.

关 键 词：三疣梭子蟹 C-型凝集素 副溶血弧菌 对虾白斑综合征病毒(WSSV) 免疫应答 

分 类 号：S968.25[农业科学—水产养殖] Q786[农业科学—水产科学]