罗氏沼虾ATG5的鉴定及其抗菌免疫功能  

Identification and Antibacterial Immunity Function of ATG5 in Macrobrachium rosenbergii

作　　者：刘洋 赵飞[2] 文菁 王玲 廖永众 邓玉婷 谭爱萍[2] 巩华[2] 赖迎迢[2] 黄志斌[2] 邵蓬 王飞飞 LIU Yang;ZHAO Fei;WEN Jing;WANG Ling;LIAO Yongzhong;DENG Yuting;TAN Aiping;GONG Hua;LAI Yingtiao;HUANG Zhibin;SHAO Peng;WANG Feifei(College of Fisheries,Tianjin Agricultural University,Tianjin 300384,China;Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences/Key Laboratory of Fishery Drug Development,Ministry Agriculture and Rural Affairs/Guangdong Provincial Key Laboratory of Aquatic Animal Immunology and Sustainable Aquaculture,Guangzhou 510380,China;Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology,Zhaoqing 526238,China)

机构地区：[1]天津农学院水产学院,天津300384 [2]中国水产科学研究院珠江水产研究所/农业农村部渔用药物创制重点实验室/广东省水产动物免疫与绿色养殖重点实验室,广东广州510380 [3]岭南现代农业科学与技术广东省实验室肇庆分中心,广东肇庆526238

出　　处：《广东海洋大学学报》2025年第1期1-11,共11页Journal of Guangdong Ocean University

基　　金：中国水产科学研究院中央级公益性科研院所基本科研业务费专项(2024SJRC8和2023TD50);广东省基础与应用基础研究基金粤穗联合基金(2023A1515110194);岭南现代农业科学与技术广东省实验室肇庆分中心项目(P20211154-0102-01);广东省农村科技特派员项目(KTP20240662)。

摘　　要：【目的】研究罗氏沼虾(Macrobrachium rosenbergii)中自噬相关基因(ATG)的免疫功能及其在抗菌免疫中的分子调节机制。【方法】从罗氏沼虾中克隆并鉴定罗氏沼虾自噬相关基因5(MrATG5),使用生物信息学方法分析MrATG5的结构特征和理化性质,采用荧光定量PCR检测MrATG5在罗氏沼虾健康组织以及受到嗜水气单胞菌(Aeromonas hydrophila)和脂多糖(Lipopolysaccharide,LPS)免疫刺激后在肝胰腺和鳃组织中的表达量,利用免疫细胞化学方法检测MrATG5蛋白在HEK 293T细胞中的亚细胞定位,通过RNA干扰技术、细菌结合和抑菌活性实验分析MrATG5的免疫功能。【结果】生物信息学分析显示,MrATG5开放阅读框(ORF)为807 bp,预测编码268个氨基酸,预测蛋白分子质量为31.04 ku,等电点为5.68,含有一个典型的自噬蛋白5(Autophagy protein 5,APG5)结构域。序列同源性分析及构建的系统进化树显示,MrATG5与日本沼虾(Macrobrachium nipponense)ATG5同源性最高,相似性为97.76%,MrATG5与甲壳类动物聚为一支,说明ATG5蛋白在甲壳类动物中进化相对保守。qRT-PCR结果显示,MrATG5在罗氏沼虾各组织中广泛表达,在肝胰腺中表达量最高(P<0.05)。MrATG5主要分布在HEK 293T细胞的细胞质中。经嗜水气单胞菌和LPS免疫刺激后,MrATG5在肝胰腺和鳃组织中的表达量均显著升高(P<0.05),表明MrATG5在罗氏沼虾抗菌免疫中发挥重要作用。经RNA干扰及嗜水气单胞菌刺激后,MrATG5可通过调控自噬参与罗氏沼虾抗菌免疫。细菌结合和抑菌活性实验发现,MrATG5原核重组表达活性蛋白可与嗜水气单胞菌、弗氏柠檬酸杆菌(Citrobacter freundii)、金黄色葡萄球菌(Staphylococcus aureus)和海豚链球菌(Streptococcus iniae)产生结合反应,并可抑制嗜水气单胞菌、弗氏柠檬酸杆菌的生长。【结论】MrATG5可通过调控自噬和抑制病原菌增殖提升罗氏沼虾的抗菌免疫力。【Objective】To study the immune function of autophagy related genes(ATG)in Macrobrachium rosenbergii and explore the specific immune regulatory mechanism of ATGs.【Method】ATG5 was identified and characterized from M.rosenbergii(MrATG5).The structural characteristics and physicochemical properties were analyzed with bioinformatics methods.The expression levels of MrATG5 in healthy tissues of M.rosenbergii and in hepatopancreas and gill tissues after immune stimulation with Aeromonas hydrophila and lipopolysaccharide(LPS)were detected by fluorescence quantitative PCR.Immunohistochemistry was used to detect the subcellular localization of MrATG5 protein in HEK 293T cells.The function of MrATG5 was analyzed by RNA interference technology,bacterial binding and antibacterial activity experiments.【Result】The open reading frame(ORF)of MrATG5 was of 807 bp encoding a peptide of 268 amino acids with a conserved autophagy protein 5(APG5)domain,with the molecular weight of 31.04 ku and an isoelectric point of 5.68.Sequence homology analysis and phylogenetic tree showed that the amino acid sequence of MrATG5 shared 97.76%similarity with that of ATG5 from Macrobrachium nipponense,and clustered with crustaceans,indicating that the ATG5 protein has evolved relatively conservatively in crustaceans.The qRT-PCR results showed that MrATG5 was widely expressed in various tissues of M.rosenbergii.MrATG5 mRNA was detected in all the examined tissues with the highest expression in hepatopancreas(P<0.05).It was found that MrATG5 was mainly distributed in the cytoplasm of HEK 293T cells.The expression level of MrATG5 mRNA in hepatopancreas and gills were significantly induced after A.hydrophila and LPS stimulation(P<0.05),which indicates that MrATG5 plays an important role in the antibacterial immunity of M.rosenbergii.MrATG5 was interfered by RNA interference and stimulated by A.hydrophila.It was found that MrATG5 could participate in the antibacterial immunity of M.rosenbergii by regulating autophagy.Additionally,the bacterial

关 键 词：罗氏沼虾 自噬相关基因5(ATG5) 嗜水气单胞菌 原核表达 抗菌免疫 

分 类 号：Q754[生物学—分子生物学] S945.41[农业科学—水产养殖]