红笛鲷IRAK-1基因克隆及哈维弧菌感染后的组织表达

Cloning and Tissue Expression of IRAK-1 Gene from Lutjanus sanguineus in Response to Vibrio harveyi Infection

用同源克隆和RACE的方法克隆红笛鲷(Lutjanus sanguineus)白细胞介素1受体相关激酶1(Interleukin-1receptor-associated kinase 1,IRAK-1)基因的cDNA全序列,并用荧光定量PCR分析其在健康鱼及哈维弧菌感染后红笛鲷的组织表达。结果表明,该序列全长3 207 bp(登录号KF728204),包含5′非编码区(UTR)202 bp,3′UTR752 bp,开放阅读框(ORF)2 253 bp,编码750个氨基酸。根据推导的氨基酸序列预测其蛋白分子质量为82.6 ku,理论等电点为6.59。IRAK-1包含1个N端死亡结构域、proST结构域、中央激酶结构域和C末端结构域。荧光定量PCR分析显示,红笛鲷IRKA-1基因在肝脏和皮肤的表达量最高,其次是心脏、鳃、肌肉和胸腺,其余组织的表达量较低。哈维弧菌侵染红笛鲷后,各组织中IRAK-1基因mRNA表达量均呈上调趋势,肝组织中变化最显著。

The full length cDNA sequence of the gene of interleukin-1receptor-associated kinase1(IRAK-1)was amplified by homologous cloning and rapid amplification of cDNA ends(RACE)from the liver of humphead snapper,Lutjanus sanguineus.The gene expression profiles in tissue of health fish and the fish infected with Vbirio harveyi were also analyzed by real time quantitative PCR(qPCR).The results showed as follows:the total cDNA sequence of humphead snapper IRAK-1was3237bp(GenBank accession number:KF728204),including5′UTR of202bp,3′UTR of782bp,and an open reading frame(ORF)of2253bp encoding750amino acids with molecule mass of82.6ku and PI of6.59.The predicted IRAK-1protein contained a N-terminal death domain(DD),a ProST domain,a central protein kinase domain(KD)and a terminus domain in structure.The real time quantitative PCR analysis showed that humphead snapper IRAK-1expressed in all examined tissues with the highest levels in liver and skin,moderate levels in heart,gill,muscle and thymus,low levels in other tissues.The mRNA expression levels of IRAK-1were significantly up-regulated in the tested tissues,especially in the liver.The results will provide a theoretical basis for further revealing the mechanism of anti-bacterial immune response of snapper IRAK-1.