吉富罗非鱼IRF9基因克隆及表达特征分析  

Cloning and expression characteristics analysis of IRF9 gene in GIFT Oreochromis niloticus

作　　者：韩鹏夫 彭文 童桂香 纪月鑫 陈铭曲 文水平 韦信贤 韦友传[1] HAN Peng-fu;PENG Wen;TONG Gui-xiang;JI Yue-xin;CHEN Ming-qu;WEN Shui-ping;WEI Xin-xian;WEI You-chuan(College of Animal Science and Technology,Guangxi University/Key Laboratory of Aquatic Animal Health Breedingand Nutrition Regulation in Guangxi Colleges and Universities,Nanning,Guangxi 530004,China;GuangxiAcademy of Fishery Sciences/Guangxi Key Laboratory of Aquatic Genetic Breeding and Health Breeding,Nanning,Guangxi 530021,China)

机构地区：[1]广西大学动物科学技术学院/广西高校水生动物健康养殖与营养调控重点实验室,广西南宁530004 [2]广西水产科学研究院/广西水产遗传育种与健康养殖重点实验室,广西南宁530021

出　　处：《南方农业学报》2024年第6期1817-1827,共11页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(32160873);广西自然科学基金项目(2023GXNSFAA026502);国家现代农业产业技术体系广西特色淡水鱼创新团队病害防控岗位功能专家项目(nycytxgxcxtd-2021-08-02)。

摘　　要：【目的】明确吉富罗非鱼(Oreochromis niloticus)干扰素调节因子9基因(OnIRF9)的结构特征和组织表达模式,以及无乳链球菌感染和Poly(I:C)刺激后的免疫应答情况,为揭示IRF9在罗非鱼免疫应答中的作用机制提供理论依据,进而丰富鱼类的IRF免疫学知识。【方法】通过RACE扩增OnIRF9基因cDNA序列,通过ExPASy、SMART、SignalP-5.0、TMHMM-2.0和Euk-mPLoc 2.0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OnIRF9基因在健康吉富罗非鱼组织中的表达特征及无乳链球菌感染和Poly(I:C)刺激后的表达变化。【结果】OnIRF9基因cDNA序列全长1828 bp,包含50 bp的5'端非编码区(5'-UTR)、485 bp的3'端非编码区(3'-UTR)和1293 bp的开放阅读框(ORF),共编码430个氨基酸残基。OnIRF9蛋白分子量为48.42 kD,理论等电点(pI)为6.23,含有IRF典型的DBD结构域和IAD结构域,不含信号肽和跨膜结构域。OnIRF9氨基酸序列与其他硬骨鱼类IRF9氨基酸序列的相似性为56.2%~95.7%,其中与美妊丽鱼IRF9氨基酸序列的相似性最高(95.7%);基于IRF9氨基酸序列相似性构建的系统发育进化树也显示,吉富罗非鱼与其他参考鱼类聚为一簇,尤其与美妊丽鱼的亲缘关系最近。OnIRF9基因在健康吉富罗非鱼脑、血液、肌肉、肝脏、皮肤、心脏、脾脏、头肾、肠道和鳃组织中均有表达,以脑组织中的相对表达量最高,显著高于其他组织(P<0.05),而鳃组织中的相对表达量最低。经无乳链球菌感染和Poly(I:C)刺激后,OnIRF9基因在吉富罗非脾脏、头肾和脑组织中的相对表达量在6 h内均极显著上升(P<0.01),但其相对表达量达到峰值的时间并不一致,提示OnIRF9基因在各组织中响应免疫应答时可能具有互补协调性。【结论】OnIRF9具有IRF典型的结构域(DBD和IAD),尤其是DBD结构域的功能在脊椎动物中较保守;OnIRF9基因在中枢神经系统中发挥重要作用,且参与吉富罗非鱼抵御病原体入�【Objective】To clarify the structural characteristics and tissue expression patterns of the interferon regula tory factor 9 gene(OnIRF9)in GIFT Oreochromis niloticus,and its immune response after infection with Streptococcus agalactis and stimulation with Poly(I:C),which provided theoretical basis for revealing the function mechanism of IRF9 in immune response reaction of GIFT O.niloticus and enriching the immunological knowledge of IRF in fish.【Method】The cDNA sequence of the OnIRF9 gene was amplified by using RACE technology,bio-informatics analysis was per formed by applying online softwares,such as ExPASy,SMART,TMHMM-2.0 and Euk-mPLoc 2.0,while real-time fluorescence quantitative PCR was used to detect the expression characterisitics of the OnIRF9 gene in healthy GIFT O.ni loticus tissues and its expression changes after infection with S.agalactiae and stimulation with Poly(I:C).【Result】The full-length cDNA sequence of OnIRF9 gene was 1828 bp,containing a 50 bp 5'-end non-coding region(5'-UTR),a 485 bp 3'-end non-coding region(UTR),and a 1293 bp open reading frame(ORF),encoding 430 amino acid residues.OnIRF9 protein had a molecular weight of 48.42 kD and a theoretical isoelectric point(pI)of 6.23 and contained two typi cal IRF domains of DBD and IAD,without signal peptides and transmembrane domains.The amino acid sequence of OnIRF9 shared 56.2%-95.7%similarity with IRF9 amino acid sequences from other teleosts fish species,had the highest similarity with IRF9 amino acid sequences of Astatotilapia calliptera(95.7%).The phylogenetic tree constructed based on IRF9 amino acid sequence similarity showed that GIFT O.niloticus clustered with other reference fish species,particu larly exhibiting the closest relationship with A.calliptera.The OnIRF9 gene was expressed in brain,blood,muscle,liver,skin,heart,spleen,head kidney,intestine and gill of healthy GIFT O.niloticus,with the highest relative expres sion level in the brain,which was significantly higher than other tissues(P<0.05),while the relative expr

关 键 词：吉富罗非鱼 干扰素调节因子9(IRF9) 无乳链球菌 Poly(I:C) 免疫应答 

分 类 号：S965.125[农业科学—水产养殖]