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

  • 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PtHMGBa。其cDNA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 kD, 理论等电点为5.94的蛋白质。PtHMGBa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, PtHMGBa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, PtHMGBa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PtHMGBa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。

     

    Abstract: 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 cDNA from P. trituberculatus (named PtHMGBa) was first cloned by using the technology of rapid amplification of cDNA ends (RACE) in our study. The full-length cDNA of PtHMGBa 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 kD. 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 PtHMGBa was mainly distributed in the haemocytes and hepatopancreas. A weak expression was detected in the eyestalk. Quantitative Real-time PCR analysis showed that PtHMGBa transcripts up-regulated significantly in haemocytes at 6h post V. Parahemolyticus inflection and up-regulated significantly in hepatopancreas at 48h post infection. Meanwhile, PtHMGBa transcripts significantly up-regulated in haemocytes at 12h post WSSV inflection, and the same result was observed in hepatopancreas. Our results suggested that PtHMGBa 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 crustaceans.

     

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