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三疣梭子蟹Toll4基因克隆及其在病原和低盐胁迫中的表达特征分析
张 杰1,2, 吕建建2,3, 刘 萍2,3, 李 健2,3, 王竹青1,2, 张小辉1,2
1.上海海洋大学水产与生命学院 上海 201306;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 青岛 266071;3.农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071
摘要:
采用RACE技术克隆得到三疣梭子蟹(Portunus trituberculatus)TLRs家族的一个新基因,将其命名为PtToll4。该基因cDNA全长为4276 bp,5¢和3¢非编码区分别为339 bp和1252 bp,编码一个含有895个氨基酸、分子量为102.5 kDa、理论等电点为6.03的蛋白质。PtToll4蛋白是一个跨膜蛋白,存在胞外区LRR、LRRCT结构域及保守的胞内区TIR结构域。同源性及系统进化分析显示,PtToll4氨基酸序列与中华绒螯蟹(Eriocheir sinensis) EsToll2的同源性最高,为61%。实时荧光定量PCR结果显示,PtToll4基因在血细胞中的表达量最高,在肝胰腺中的表达量最低。利用副溶血弧菌(Vibrio parahemolyticus)和对虾白斑综合征病毒(WSSV)对三疣梭子蟹进行注射感染,结果显示,经WSSV感染后,PtToll4基因在血细胞中的表达量显著提高,在6 h时出现峰值,为对照组的5.03倍(P<0.01)。经副溶血弧菌感染后,PtToll4基因仅在48 h略微出现上调,其余时间点与对照组无显著差异。由此推测,PtToll4基因在三疣梭子蟹应答WSSV的免疫过程中发挥了重要功能。此外发现,低盐胁迫显著抑制了PtToll4基因在三疣梭子蟹血细胞中的表达,这可能是导致低盐环境下三疣梭子蟹等甲壳类动物免疫力下降的原因之一。由此推测,PtToll4基因在三疣梭子蟹先天免疫过程中发挥了重要功能,本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研究提供了数据支持。
关键词:  三疣梭子蟹  PtToll4  基因克隆  先天免疫  表达分析
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Cloning of Toll4 in Portunus trituberculatus and Its Expression in Responding to Pathogenic Infection and Low Salinity Stress
ZHANG Jie1,2, LÜ Jianjian2,3, LIU Ping2,3, LI Jian2,3, WANG Zhuqing1,2, ZHANG Xiaohui1,2
1.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306;2.Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071;3.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071
Abstract:
Portunus trituberculatus, an economically important food species in China, lacks adaptive immunity and can induce effective immune responses to resist pathogen via innate immunity. Toll-like receptors as pattern recognition receptors (PRRs) play crucial role in innate immune responses. The current study found a novel Toll gene named as PtToll4 from P. trituberculatus by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of PtToll4 was 4276 bp, which contains an open reading frame (ORF) of 2685 bp, a 3¢ untranslated region (UTR) of 1252 bp, and a 5¢ UTR of 339 bp. The ORF encoded a polypeptide of 895 amino acids with a predicted molecular weight of 102.5 kDa. PtToll4 is a transmembrane protein that contains several common structural domains of TLRs, such as LRR, LRRCT motif in the extracellular domain and a TIR domain in the intracellular region. The BLAST analysis showed that PtToll4 shared the highest homology with Eriocheir sinensis Toll2. PtToll4 was widely expressed in hemocyte, heart, gill, hepatopancreas, stomach, intestine, muscle, cuticle, and eyestalk, and mainly distributed in the hemocyte and cuticle. A weak expression level was detected in the hepatopancreas. WSSV inflection significantly induced PtToll4 expression in hemocyte at 6 h, and V. parahemolyticus inflection had a little effect on it only at 48 h. Low salinity reduced PtToll4 expression. Our results suggested that PtToll4 may play an important role in innate immune of P. trituberculatus and would provide useful information for the research of immunity regulation in crustaceans.
Key words:  Portunus trituberculatus  PtToll4  Gene cloning  Innate immune  Expression analysis