三疣梭子蟹HMGBa基因克隆及其应答不同病原入侵的表达特征  

CLONING OF HMGBA IN PORTUNUS TRITUBERCULATUS AND ITS EXPRESSION IN RESPONDING TO BACTERIAL AND VIRAL INFECTIONS

作　　者：张杰[1,2] 吕建建[1,3] 刘萍[1,3] 李健 王竹青[1,2] 张小辉 

机构地区：[1]中国水产科学研究院黄海水产研究所,农业部海洋渔业可持续发展重点实验室,青岛266071 [2]上海海洋大学水产与生命学院,上海201306 [3]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,青岛266071

出　　处：《水生生物学报》2017年第6期1193-1199,共7页Acta Hydrobiologica Sinica

基　　金：泰山领军人才工程高效生态农业创新类计划(LJNY2015002);国家自然科学基金面上项目(41506186);海洋渔业科学与食物产出过程功能实验室开放课题(2016LMFS-A12)资助~~

摘　　要：为研究三疣梭子蟹(Portunus trituberculatus)高迁移率族蛋白B(High-mobility group box protein,HMGB)在其先天免疫中发挥的功能,利用RACE技术首次克隆得到了三疣梭子蟹HMGBa基因,命名为Pt HMGBa。其c DNA序列全长1030 bp,其中5′端非编码区(UTR)为94 bp,3′端非编码区(UTR)为255 bp,开放阅读框(ORF)为681 bp,编码一个含有227个氨基酸,分子量25.82 k D,理论等电点为5.94的蛋白质。Pt HMGBa蛋白包含2个HMG盒结构域和一个酸性尾部结构域。分析表明,三疣梭子蟹HMGBa氨基酸序列与凡纳滨对虾(Litopenaeus vannamei)HMGBa相似度最高。实时荧光定量PCR结果显示,Pt HMGBa基因在血细胞和肝胰腺的表达量最高,在眼柄中表达量最低。在副溶血弧菌和WSSV感染过程中,Pt HMGBa基因在肝胰腺和血细胞中均出现了表达上调。其中,经副溶血弧菌感染后,该基因在上述2种组织中分别于48h和6h达到表达量的峰值;经WSSV感染后,该基因在2种组织中均在12h达到表达量的峰值。结果表明Pt HMGBa基因参与了三疣梭子蟹抵御外来病原的免疫响应,研究为深入开展三疣梭子蟹和其他甲壳动物的免疫调控机理提供了科学依据。Portunus trituberculatus is an economically important aquaculture species in China. As crustacean, it is lack of adaptive immunity and can induce effective immune responses to resist pathogen relying on innate immunity. Vibrio parahemolyticus and white spot syndrome virus（WSSV） have caused significant economic damage to P. trituberculatus aquaculture. High mobility group box（HMGB） proteins are known to be involved in diverse functions in mammalian cells. In crustacean, very limited studies on HMGB proteins have been documented. To investigate the role of high mobility group box（HMGB） proteins in innate immune system of P. trituberculatus, the HMGB homologue c DNA from P. trituberculatus（named Pt HMGBa） was first cloned by using the technology of rapid amplification of c DNA ends（RACE） in our study. The full-length c DNA of Pt HMGBa was 1030 bp in length, which includes an open reading frame（ORF） of 681 bp, a 255 bp 3′ untranslated region and a 94 bp 5′ untranslated region. The ORF encoded a polypeptide of 227 amino acids with a predicted molecular weight of 25.82 k D. The polypeptide contained two HMG boxes domain and a C-terminal acidic tail composed of 24 rich Asp/Glu residues. The BLAST analysis showed that HMGB shared the highest homology with Litopenaeus vannamei HMCBa. The expression of Pt HMGBa was mainly distributed in the haemocytes and hepatopancreas. A weak expression was detected in the eyestalk. Quantitative Real-time PCR analysis showed that Pt HMGBa transcripts up-regulated significantly in haemocytes at 6 h post V. Parahemolyticus inflection and up-regulated significantly in hepatopancreas at 48 h post infection. Meanwhile, Pt HMGBa transcripts significantly up-regulated in haemocytes at 12 h post WSSV inflection, and the same result was observed in hepatopancreas. Our results suggested that Pt HMGBa may play important roles in innate immunity of the crab, and would provide useful information for the research of immune regulation in P. trituberculatus and other crustace

关 键 词：三疣梭子蟹 高迁移率蛋白B 基因克隆 先天免疫 表达分析 

分 类 号：S917[农业科学—水产科学]