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
刘洋,赵飞,文菁,王玲,廖永众,邓玉婷,谭爱萍,巩华,赖迎迢,黄志斌,邵蓬,王飞飞
摘要(Abstract)：
【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（Citrobacterfreundii）、金黄色葡萄球菌（Staphylococcusaureus）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。
关键词(KeyWords)： 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
基金项目(Foundation): 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）
作者(Author): 刘洋,赵飞,文菁,王玲,廖永众,邓玉婷,谭爱萍,巩华,赖迎迢,黄志斌,邵蓬,王飞飞
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