尼罗罗非鱼选择性自噬接头受体基因(sqstm1)克隆及其功能分析  

Cloning and Functional Analysis of Sequestosome 1(sqstm1)Gene in Nile Tilapia(Oreochromis niloticus)

作　　者：黄美玲 江栢坚 冯佳敏 李星 黄瑜[1] 简纪常[1] HUANG Mei-Ling;JIANG Bai-Jian;FENG Jia-Min;LI Xing;HUANG Yu;JIAN Ji-Chang(College of Fisheries/Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy culture/Key Laboratory of Control for Disease of Aquatic Economic Animals of Guangdong Higher Education Institutes,Guangdong Ocean University,Zhanjiang 524088,China)

机构地区：[1]广东海洋大学水产学院/广东省水产动物病害防控与健康养殖重点实验室/广东省高等学校水生经济动物疾病控制重点实验室,湛江524088

出　　处：《农业生物技术学报》2023年第3期569-579,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32073006);广东省自然科学基金(2022A1515010553)。

摘　　要：尼罗罗非鱼(Oreochromis niloticus)选择性自噬接头受体蛋白1(sequestosome 1 receptor protein,Sqstm1)是一种选择性自噬接头蛋白,可选择性参与清除细胞内受损的细胞器和蛋白质。为分析sqstm1基因的功能,研究其在健康尼罗罗非鱼各组织中的表达分布及抗菌抗病毒应答过程中的表达模式,本研究克隆了On-sqstm1基因(GenBank No.XP_005463852)ORF序列,通过生物信息学及亚细胞定位分析Onsqstm1基因的序列和定位特征,利用qPCR技术分析其在健康罗非鱼各组织及不同刺激物刺激后各组织中的表达模式,采用双荧光素酶报告系统检测On-sqstm1基因过表达对核因子κB(nuclear factor kappa-B,NF-κB)活性的影响。结果显示,本研究成功克隆了On-sqstm1基因的ORF序列,长度为1287 bp,编码428个氨基酸;亚细胞定位结果显示On-Sqstm1蛋白定位于细胞质。qPCR结果显示On-sqstm1基因在罗非鱼各组织中均有表达,在肝脏中表达量最高;在无乳链球菌(Streptococcus agalctiae)和Poly I:C病毒类似物刺激后,该基因在罗非鱼的脑、头肾、肝脏、肠道及脾脏组织中的表达量均显著上调(P<0.05)。双荧光素酶报告系统显示On-Sqstm1蛋白可以显著激活NF-κB活性。以上结果显示,On-Sqstm1蛋白可能通过介导NF-κB信号通路的活性参与对病原体感染的免疫应答过程。本研究为进一步研究On-Sqstm1蛋白的作用机制提供了参考。Sequestosome 1 receptor protein(Sqstm1)is a selective autophagic adaptor protein in Nile tilapia(Oreochromis niloticus),which selectively participates in the removal of intracellular damaged organelles and proteins.To analyse the function of sqstm1 gene,and expression distribution of this gene in healthy Nile tilapia tissues and its expression pattern in the process of resisting bacterium and virus.In this study,the ORF of the gene On-sqstm1(GenBank No.XP_005463852)was cloned,and the characteristics of bioinformatics and the subcellular localization were analysed,its expression patterns in healthy Nile tilapia tissues and after stimulation with different stimulant were analyzed by qPCR.The effect on nuclear factor kappa-B(NF-κB)signal path of On-sqstm1 was examined by using the dual luciferase reporting system.The results revealed that the ORF of the gene On-sqstm1 was successfully cloned and identified.which was 1287 bp in length and encoded 428 amino acids.The results of subcellular localization showed that On-Sqstm1 was localized in the cytoplasm,the qPCR results revealed that On-sqstm1 was expressed in all tissues of tilapia and the highest in the liver.After challenged with the inactivated Streptococcus agalctiae and Poly I:C virus analogues,the expressions of brain,head kidney,liver,intestine and spleen significantly increased(P<0.05).The results of dual luciferase reporting system revealed that On-Sqstm1 significantly activated the NF-κB signal pathway activity.The above results indicated that On-Sqstm1 protein may participate in the immune response to pathogen infection by mediating NF-κB signaling pathway activity.This study provides a reference for further research on the mechanism of On-Sqstm1.

关 键 词：尼罗罗非鱼 自噬接头受体蛋白1(Sqstm1) 无乳链球菌 PolyⅠ:C 表达模式 

分 类 号：S943[农业科学—水产养殖]