斜带石斑鱼BAG-1基因克隆及表达分析

Cloning and expression analysis of BAG-1 gene in Epinephelu coioides

  • 摘要: 【目的】明确斜带石斑鱼(Epinephelu coioidesBAG-1基因(EcBAG-1)的结构特征、组织表达分布及在脂多糖(LPS)和聚肌胞苷酸(PolyI:C)刺激后的表达变化,进一步揭示鱼类BAG-1在病原感染中的作用机制,也为了解鱼类的抗病免疫提供参考依据。【方法】依据EcBAG-1基因EST序列设计特异性引物克隆EcBAG-1基因,通过SMART、TMHMM Server v.2.0、SOPMA、SWISS-MODEL、ExPASy、ClustalX及MEGA 5.0等在线软件进行生物信息学分析,并采用实时荧光定量PCR检测EcBAG-1基因的组织表达特征及其在LPS和PolyI:C刺激后的表达模式。【结果】克隆获得的EcBAG-1基因开放阅读框(ORF)长624 bp,共编码207个氨基酸残基;EcBAG-1蛋白分子量为49.84 kD,理论等电点(pI)为5.18,为疏水性蛋白。EcBAG-1蛋白具有BAG-1家族保守的BAG结构域和UBQ泛素相关结构域,无跨膜结构域,主要分布在细胞质和细胞核,其二级结构中α-螺旋占65.22%、延伸链占10.63%、无规则卷曲占20.29%、β-转角仅占3.86%。EcBAG-1氨基酸序列与大黄鱼的BAG-1氨基酸序列相似性最高(96.62%),亲缘关系最近;与原鸡和热带爪蛙的亲缘关系相对较远,对应的BAG-1氨基酸序列相似性分别为58.45%和57.50%。EcBAG-1基因在斜带石斑鱼的肝脏、胸腺、脾脏、头肾、鳃、皮肤、肌肉、大脑、外周血淋巴细胞及心脏等组织中均有表达,且以在大脑中的相对表达量最高,显著高于在其他组织中的相对表达量(P>0.05,下同);经LPS和PolyI:C刺激24 h后,斜带石斑鱼脾脏中的EcBAG-1基因相对表达量显著上调。【结论】EcBAG-1蛋白含有BAG-1蛋白家族典型的BAG结构域和UBQ结构域,具有与哺乳动物BAG-1相似的生物学功能,即EcBAG-1基因在斜带石斑鱼抵御细菌/病毒感染的过程中发挥重要作用,为深入了解鱼类的抗感染机理拓宽了思路。

     

    Abstract: 【Objective】To investigate the structural characteristics,tissue expression distribution of BAG-1 gene (EcBAG-1)in Epinephelu coioides and its expression changes of it after stimulation of lipopolysaccharide(LPS)and polyinosinic acid(PolyI∶C),with an aim to further reveal the mechanism of fish BAG-1 in pathogen infection,so as to provide a reference for understanding the immunity of fish against disease.【Method】The EcBAG-1 gene was cloned by designing primers based on its EST sequence,and bioinformatics analysis was performed by using online software such as SMART,TMHMM Server v.2.0,SOPMA,SWISS-MODEL,ExPASy,ClustalX and MEGA 5.0. The tissue expression characteristics of EcBAG-1 gene and its expression pattern after LPS and Poly I:C stimulation were detected by using realtime fluorescence quantitative PCR.【Result】The open reading frame(ORF)of the cloned EcBAG-1 gene was 624 bp in length,encoding 207 amino acids,and EcBAG-1 protein molecular weight was 49.84 kD,theoretical isoelectric point (pI)was 5.18 and it was a hydrophobic protein. Ec-BAG-1 protein had a conserved BAG domain and an UBQ ubiquitinrelated domain of the BAG-1 family,and no transmembrane domain,it mainly existed in the cytoplasm and nucleus. In the secondary structure,α - helix accounted for 65.22%,extended chain 10.63%,irregular coiled coil 20.29% and β - turned angle only 3.86%. EcBAG-1 amino acid sequence of E. coioides had the highest similarity(96.62%)with Larimichthys crocea,and the two species was the most closely related. E. coioides was relatively distantly related to Gallus gallus and Xenopus tropicalis,with corresponding BAG-1 amino acid sequence similarities of 58.45% and 57.50%,respectively. EcBAG-1 gene was expressed in liver,thymus,spleen,head kidney,gill,skin,muscle,brain,peripheral blood lymphocytes and heart of E. coioides,and the relative expression was highest in brain,which was significantly higher than that in other tissues(P>0.05,the same below). After 24 h of LPS and PolyI:C stimulation,relative expression of EcBAG-1 gene in the spleen of E. coioides.【Conclusion】EcBAG-1 protein contains the typical BAG structural domain and UBQ structural domain of the BAG-1 protein family,and it has biological function similar to mammalian BAG-1 of mammals,which means EcBAG-1 gene plays an important role in the resistance to bacterial/viral infection in E. coioides, broadening the idea of understanding the anti-infection mechanism in fish.

     

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