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

Cloning of Toll4 in Portunus trituberculatus and Its Expression in Responding to Pathogenic Infection and Low Salinity Stress

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Pt Toll4。该基因c DNA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 k Da、理论等电点为6.03的蛋白质。Pt Toll4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,Pt Toll4氨基酸序列与中华绒螯蟹(Eriocheir sinensis)Es Toll2的同源性最高,为61%。实时荧光定量PCR结果显示,Pt Toll4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,Pt Toll4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,Pt Toll4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,Pt Toll4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Pt Toll4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,Pt Toll4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。

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.