红笛鲷Integrin-β基因的克隆及表达分析

Cloning and Expression Analysis of Integrin-β Gene in Lutjanus sanguineus

为了探讨鱼类Integrin-β的组织分布及其在病原刺激后的表达模式,本研究以中国南方主要海水养殖品种-红笛鲷为研究对象,根据实验室构建红笛鲷cDNA差减文库中的EST片段,设计引物,通过RACE技术得到红笛鲷Integrin-β基因的cDNA全长(GenBank登录号:MG792792,命名为Ls-Integrin-β),该基因全长3 976 bp,开放阅读框(ORF)包含2 409 bp碱基,编码802个氨基酸,理论分子量大小为88.99 kD。氨基酸推导序列分析发现Ls-Integrin-β具有整合素家族保守结构域,C端存在一个跨膜结构域。系统进化树分析红笛鲷与鰤Integrin-β相似度最高。Ls-Integrin-β在健康红笛鲷体内的多种组织均有表达,其中肌肉表达量最高,脑、体肾、皮肤其次,肠道表达量最低。哈氏弧菌刺激后,红笛鲷脾脏中Integrin-β的表达在感染后6 h显著性(p<0.05)上调,在12 h达到顶峰。以上结果表明,Integrin-β可能参与了宿主抵御病原入侵的免疫反应,为进一步了解红笛鲷抗菌免疫提供理论基础。

In order to investigate the tissue distribution of fish Integrin-β and its expression pattern after pathogenic stimulation, taking the main mariculture species of the South China, Red snapper(Lutjanus sanguineus), as the research object, in this study, we designed primers based on EST sequence in Lutjanus sanguineus subtractive cDNA library of our laboratory, the Integrin-β was obtained from Lutjanus sanguineus spleen using RACE PCR(GenBank accession number: MG792792;designated as Ls-Integrin-β). The full length cDNA of Ls-Integrin-βwas 3 976 bp, contained an ORF of 2 409 bp encoding a polypeptide of 802 amino acids with predicted molecular of 88.99 k D. Amino acid sequence showed that Ls-Integrin-β possesses a typical integrin family domain, and a transmembrane domain in C terminal. Phylogenetic tree analysis indicated that the highest similarity was between Ls-Integrin-β and Seriola dumerili Integrin-β. Ls-Integrin-β was detected in all chosen tissues by qRT-PCR with the highest expression in muscle, brain, kidney and skin followed, the lowest in intestine. After Vibrio harveyi challenge, the expression level of Ls-Integrin-β in spleen was up-regulated significantly(p<0.05) at 6 h, peaked at 12 h. These results indicated that Integrin-β played an important role in host defense against pathogen invasion,provided a theory basis for studying anti-bacterial immunity of Lutjanus sanguineus.