克氏原螯虾自噬相关基因PcAtg2的克隆及其在白斑综合征病毒胁迫下的表达分析  

Cloning and Expression Analysis of the Autophagy Related Gene PcAtg2 in Procambarus clarkii Under White Spot Syndrome Virus Stress

作　　者：祝孟茹 问露洁 占铭 公洁 席昌俊 闻海波[1,2] 沈怀舜 ZHU Mengru;WEN Lujie;ZHAN Ming;GONG Jie;XI Changjun;WEN Haibo;SHEN Huaishun(Wuxi Fisheries College,Nanjing Agricultural University,Wuxi 214081,China;Key Laboratory of Integrated Rice-Fish Farming Ecology,Ministry of Agriculture and Rural Affairs,Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi 214081,China)

机构地区：[1]南京农业大学无锡渔业学院,江苏无锡214081 [2]中国水产科学研究院淡水渔业研究中心农业农村部稻鱼综合养殖生态重点实验室,江苏无锡214081

出　　处：《渔业科学进展》2023年第5期137-152,共16页Progress in Fishery Sciences

基　　金：江苏省自然科学基金(BK20181138);中央级公益性科研院所基本科研业务费专项(2019JBFZ09)共同资助。

摘　　要：为了解自噬相关基因Atg2在克氏原螯虾(Procambarus clarkia)先天免疫中的作用,本研究克隆了克氏原螯虾Atg2(PcAtg2)基因全长序列。生物信息学分析显示,PcAtg2蛋白编码序列全长为9966 bp,推测其编码2189个氨基酸。组织定量表达分布显示,PcAtg2在克氏原螯虾的各个组织中均有表达,其中在肝胰腺中表达最高,在眼柄中表达最低。在白斑综合征病毒(WSSV)感染实验中,PcAtg2基因表达量在不同组织中均呈现显著上调趋势。RNA干扰(RNAi)实验显示,PcAtg2基因沉默后,WSSV在克氏原螯虾体内的增殖明显被抑制,同时,自噬相关基因的表达量上调。透射电镜分析结果显示,在WSSV感染后,PcAtg2基因沉默组中克氏原螯虾肝胰腺组织中的自噬小体多于对照组。本研究结果可为了解克氏原螯虾应对WSSV胁迫下的调控机制提供理论参考。Procambarus clarkii is commonly known as crayfish and has become one of the main species of freshwater aquaculture in China because of its delicious meat and strong adaptability to the environment.The incredible demand promotes the rapid development of the crayfish breeding industry.Viral diseases caused by white spot syndrome virus(WSSV)are widely spread in crustaceans,including P.clarkii.WSSV has become a serious threat to the crayfish breeding industry because of its extremely fast transmission and associated high mortality.Virus infection can directly induce autophagy mechanisms.Autophagosomes can wrap virus particles and transport them to lysosomes for degradation.As a highly conserved cellular defense mechanism,autophagy plays an important role in the regulation of virus infections.However,many viruses have evolved special mechanisms to resist autophagy regulation or use the membrane structure produced by autophagy body formation to complete their own replication.In this study,WSSV in susceptible P.clarkii were explored to determine how autophagy related genes of P.clarkii participate in the regulation of virus infection.To study the role of the autophagy related gene(Atg2)in the innate immunity of P.clarkii,the full-length sequence of the Atg2 gene in P.clarkii(named PcAtg2)was cloned using the total RNA of P.clarkii hepatopancreas as a template with the rapid-amplification of cDNA ends technique(RACE).The bioinformatic analysis showed that the total length of the PcAtg2 gene sequence in P.clarkii was 9966 bp,including a 582 bp 5'non coding region,2817 bp 3'non coding region,and 6567 bp open reading frame.We speculate it encodes 2189 amino acids.Multiple sequence alignments showed the PcAtg2 gene had the characteristic sequence of the Atg family,with 65 serine phosphorylation sites,and 48 glycosylation sites.The amino acid sequence of PcAtg2 in P.clarkii had the highest homology with the Homarus americanus Atg2 gene.The distribution of the PcAtg2 gene in the gill,heart,midgut,hepatopancreas,stomach,muscle,

关 键 词：克氏原螯虾 Atg2 基因克隆 WSSV RNA干扰 

分 类 号：S966.12[农业科学—水产养殖]