虾夷马粪海胆TLR基因cDNA克隆及表达分析

采用同源克隆和cDNA末端快速扩增（ RACE）技术克隆获得一条虾夷马粪海胆Strongylocentrotus in-termedius TLR基因的全长cDNA序列SiTLR-1（GenBank： HQ259110）,并采用实时定量PCR技术分析了其在组织中的分布以及在致病弧菌Vibrio fortis、β-D葡聚糖和dsRNA刺激后的表达情况。结果表明： SiTLR-1全长序列为3637 bp,形成16个α螺旋、33个β折叠； SiTLR-1蛋白有两个跨膜区段,在725-750位有跨膜区,发现1个信号肽（1-32aa）、1个亮氨酸重复富集区（LRR）； SiTLR-1在检测的各组织中均有表达,在体腔液中表达量显著高于围口膜、管足和齿间肌（ P〈0.05）；经V. fortis、β-D-葡聚糖刺激后,体腔液中的SiTLR-1表达量显著上升,刺激后12 h达到最高值；经dsRNA刺激后,体腔液中的SiTLR-1表达变化较小,仅在3 h 时略有升高。研究表明, TLR 基因参与虾夷马粪海胆的免疫应答,克隆获得的SiTLR-1可特异性识别细菌和β-D-葡聚糖。

Cloning and expression analysis of TLR cDNAfrom sea urchin Strongylocentrotus intermedius

One complete cDNA of TLR named SiTLR-1 （ GenBank：HQ259110 ） was cloned from sea urchin Strongylocentrotus intermedius through degenerate primer PCR amplification and SmartTM RACE technology. Expres-sion distribution in different tissue after challenge with Vibrio fortis,β-D glucosan,and dsRNA were assessed using quantitative real-time PCR （ qRT-PCR） . The full-length cDNA sequence of SiTLR-1 is 3637 bp,with 16 α-he-lix,33 β-sheet;composed of 1 transmembrane domain （725-750aa）,a putative signal peptide （1-32aa）, and a leucine-rich repeat （ LRR） . SiTLR-1 was assessed in all tested tissues （ peristomial membrane,gut,coelomic fluid, muscle in Aristotles lantern,tube feet） ,the expression level in coelomic fluid was significantly higher than the others （ P〈0 . 05 ）; the expression level of SiTLR-1 in coelomic fluid was strongly up-regulated after challenge with V. fortis and β-D glucosan,reach the highest point at 12 h;the expression level of SiTLR-1 showed no significant difference after challenge with dsRNA, just weakly up-regulated at 3 h. The findings showed that, TLR genes par-ticipated in the immune response of sea urchin S. intermedius, SiTLR-1 can specifically identify bacteria andβ-D glucosan.