CAJ | 학술논문

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。
채용동원극륭화cDNA말단쾌속확증( RACE)기술극륭획득일조하이마분해담Strongylocentrotus in-termedius TLR기인적전장cDNA서렬SiTLR-1(GenBank： HQ259110),병채용실시정량PCR기술분석료기재조직중적분포이급재치병호균Vibrio fortis、β-D포취당화dsRNA자격후적표체정황。결과표명： SiTLR-1전장서렬위3637 bp,형성16개α라선、33개β절첩； SiTLR-1단백유량개과막구단,재725~750위유과막구,발현1개신호태(1~32aa)、1개량안산중복부집구(LRR)； SiTLR-1재검측적각조직중균유표체,재체강액중표체량현저고우위구막、관족화치간기( P<0.05)；경V. fortis、β-D-포취당자격후,체강액중적SiTLR-1표체량현저상승,자격후12 h체도최고치；경dsRNA자격후,체강액중적SiTLR-1표체변화교소,부재3 h 시략유승고。연구표명, TLR 기인삼여하이마분해담적면역응답,극륭획득적SiTLR-1가특이성식별세균화β-D-포취당。
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.