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

作　　者：李星 吴子杰 温依铭 刘结红 蔡佳[1,2,3] 简纪常 LI Xing;WU Zi-jie;WEN Yi-ming;LIU Jie-hong;CAI Jia;JIAN Ji-chang(Fisheries College,Guangdong Ocean University,Zhanjiang,Guangdong 524088,China;Guangdong Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals,Zhanjiang,Guangdong 524088,China;Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes,Zhanjiang,Guangdong 524088,China)

机构地区：[1]广东海洋大学水产学院,广东湛江524088 [2]广东省水产经济动物病原生物学及流行病学重点实验室,广东湛江524088 [3]广东省水产经济动物病害控制重点实验室,广东湛江524088

出　　处：《南方农业学报》2022年第5期1399-1406,共8页Journal of Southern Agriculture

基　　金：广西自然科学基金项目(2020GXNSFAA297243);广西红树林滨海湿地生态保护与可持续利用人才小高地人才培育项目(BGMRC202101);广东省高校“海之帆—起航计划”大学生科技创新项目(qhjhzr201820);广东海洋大学“冲一流”专项(231419013)。

摘　　要：【目的】明确斜带石斑鱼(Epinephelu coioides)BAG-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。【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%andβ-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

关 键 词：斜带石斑鱼 BAG-1基因 病原感染 免疫反应 表达分析 

分 类 号：S965.334[农业科学—水产养殖]