半滑舌鳎甘露糖结合凝集素相关丝氨酸蛋白酶1基因的克隆及表达分析

Cloning and Expression Analysis of the MASP1 Gene from the Half-Smooth Tongue Sole(Cynoglossus semilaevis)

甘露糖结合凝集素相关丝氨酸蛋白酶1(Mannan-binding lectin associated serine protease 1,MASP1)是补体凝集素途径中起重要作用的激活蛋白。本研究以半滑舌鳎(Cynoglossus semilaevis)为研究对象,应用RACE技术和实时荧光定量qRT-PCR技术对半滑舌鳎MASP1基因(Cynoglossus semilaevis MASP1,CsMASP1)进行了克隆和表达模式分析。结果显示,CsMASP1基因c DNA序列全长为2507 bp,5′非编码区长82 bp,3′非编码区长142 bp,开放阅读框长2283 bp,共编码760个氨基酸;CsMASP1基因包含13个外显子和12个内含子,与多数已知鱼类的MASP1基因结构一致;SMART分析显示,CsMASP1包含6个结构域,与哺乳动物、鸟类、其他鱼类的结构域一致;同源比对发现,CsMASP1和鱼类的相似度较高,与金目鲈(Latescalcarifer)的相似性最高,为76%。CsMASP1基因在11种健康组织(血液、脑、鳃、性腺、心脏、头肾、肠、肝、皮肤、脾和后肾)中均有表达,其中,在肝、脾和头肾中表达量较高;鳗弧菌(Vibro anguillarum)感染后,CsMASP1基因在6种免疫组织(血液、鳃、头肾、肠、肝和脾)中呈现不同的表达模式,在6种免疫组织中呈现明显的上调表达,肝的表达峰值出现在感染后24 h;脾和鳃的表达峰值出现在感染后6 h;肠、头肾和血液的表达峰值均出现在感染后12h;随着病原菌感染时间增加,基因表达量逐渐降低并恢复至正常水平。研究表明,CsMASP1基因参与了半滑舌鳎的免疫应答过程,本结果为半滑舌鳎的免疫机理研究奠定了基础。

Mannan-binding lectin-associated serine protease 1(MBL associated serine protease 1,MASP1)is an important activator in the complement lectin pathway.In this study,the cDNA of the MASP1 gene from Cynoglossus semilaevis(CsMASP1)was cloned using a RACE method based on the partial sequence of CsMASP1,and gene expression was performed by qRT-PCR.The results showed that the full length of cDNA was 2507 bp in size,including a 5′-untranslated region(UTR)of 82 bp,3′-UTR of 142 bp,and a complete open reading frame(ORF)of 2283 bp,encoding 760 amino acids.The theoretical isoelectric point(pI)of the predicted protein was 5.33 and the molecular weight was 84.95 kDa.Homologous alignment showed that the amino acids sequence of CsMASP1 had a high identity with those of other species,approximately 42%~76%.The CsMASP1 gene was expressed in all tested tissues(liver,intestine,spleen,head-kidney,gill,blood,brain,skin,heart,trunk-kidney,and gonad)in the healthy C.semilaevis and the highest expression was in the liver(185.4).To study the expression patterns of the CsMASP1 gene in an immune response,the specific expression of CsMASP1 was performed after Vibrio anguillarum infection.The results showed that the expression of the CsMASP1 gene was up-regulated in the liver,gill,blood,intestine,head-kidney,and spleen after V.anguillarum infection.The most significantly up-regulated expression and the peak level at 6 h reached 15.4 times baseline in the spleen.The results indicate that the CsMASP1 genes are involved in the immune response.