罗氏沼虾RXR基因克隆及其对蜕皮相关基因的调控  

Cloning and effect of RXR on molting-related genes expression in Macrobrachium rosenbergii

作　　者：姜群 高炜峰 许文静 纪鹏 顾舒文 张颖杰 高晓建 张晓君[1] JIANG Qun;GAO Weifeng;XU Wenjing;JI Peng;GU Shuwen;ZHANG Yingjie;GAO Xiaojian;ZHANG Xiaojun(College of Animal Science and Technology,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学动物科学与技术学院,江苏扬州225009

出　　处：《水产学报》2024年第8期53-64,共12页Journal of Fisheries of China

基　　金：国家自然科学基金(32002386);江苏省种业振兴揭榜挂帅项目[JBGS(2021)120];江苏现代农业产业技术体系建设专项[JATS(2021)505]。

摘　　要：为研究维甲酸类X受体(RXR)在甲壳动物蜕皮过程中的调控作用,实验采用RACE技术克隆获得罗氏沼虾Mr-RXR基因,通过实时荧光定量PCR(qRT-PCR)分析了Mr-RXR及其他蜕皮相关基因(MIH、EcR、E75和CHIT)在罗氏沼虾不同组织、不同蜕皮阶段的表达情况,并采用RNA干扰(RNAi)探究Mr-RXR对罗氏沼虾蜕皮相关基因表达的调控作用。结果显示,Mr-RXR基因cDNA全长2241 bp(GenBank登录号MZ501612),包括36 bp的5'末端非翻译区(UTR)和719 bp的3'UTR,开放阅读框(ORF)为1386 bp,共编码461个氨基酸。罗氏沼虾RXR氨基酸序列与甲壳动物同源性较高,存在保守的DNA结合域(DBD)和配体结合域(LBD)。Mr-RXR在罗氏沼虾所有组织中均表达,精巢、眼柄、心脏等组织中表达量较高,其表达水平随蜕皮周期改变,眼柄和肌肉组织中均在蜕皮前期表达量最高,与EcR基因表达模式基本同步;E75和CHIT基因表达模式较为相似,眼柄和肌肉组织分别在蜕皮前期和蜕皮后期高表达;MIH基因仅在眼柄组织表达,且蜕皮间期表达量最高。采用3μg dsRNA/g虾的剂量进行为期12 d的RNAi实验诱导Mr-RXR沉默,罗氏沼虾出现大量死亡,qRT-PCR显示,眼柄和肌肉中Mr-RXR干扰效率分别为79%和55%。眼柄组织中Mr-RXR基因沉默后,MIH基因表达未受影响,EcR、E75和CHIT基因表达水平显著降低,肌肉组织中,Mr-RXR沉默仅造成CHIT基因表达水平的显著下降,推测Mr-RXR可能通过影响EcR、E75和CHIT基因表达调控罗氏沼虾蜕皮过程。本研究结果将为甲壳动物蜕皮调控机制研究提供基础资料。Like all arthropods,crustaceans shed the exoskeleton periodically through their life cycle,which is called ecdysis.Molting occurs throughout the life cycle of crustaceans and significantly influences their growth,reproduction,and survival.This physiological event is regulated by several hormones,in which ecdysteroids are the important part.The effect of ecdysteroids is mediated by a heterodimer of ecdysone receptor(EcR)and retinoid X receptor(RXR)homolog,ultraspiracle(USP)in insects or RXR in crustaceans.EcR/RXR or EcR/USP complex could bind to response elements to elicit expression of early ecdysone responsive genes,such as E75,and eventually to regulate downstream transcriptional ecdysteroid cascade reaction.RXR,belonging to a nuclear receptor family,plays an important role in the regulation of molting.In recent years,RXR has been identified in several crustaceans,such as Eriocheir sinensis,Callinectes sapidu,Gecarcinus lateralis,Leptuca pugilator,Macrobrachium nipponense and so on.However,most of the studies focused on the cloning and specific expression analysis among different tissues or different molting stages,its regulation of other molting-related genes remains unclear.Therefore,this study identified a retinoid X receptor(RXR)in M.rosenbergii termed Mr-RXR and further investigated its regulation function through its mRNA expression analysis at different molting stages and RNA interference(RNAi).The full length of the Mr-RXR was 2241 bp,consisting of a 36 bp 5'UTR(untranslated regions),a 719 bp 3'-UTR,and a 1386 bp ORF(open reading frame).It encoded 461 amino acids with a predicted molecular weight of 50.6 ku and the theoretical isoelectric point of 7.02.Mr-RXR showed high similarity to other crustaceans with conserved DNA binding domain(DBD)and ligand binding domain(LBD).Phylogenetic analyses showed that the RXR of crustaceans clustered in a group,with the closest relationship between M.rosenbergii and M.nipponense.The expression of Mr-RXR was detected in 11 tissues of M.rosenbergii,including the stomach

关 键 词：罗氏沼虾 RXR基因 蜕皮 表达分析 RNA干扰(RNAI) 

分 类 号：Q786[生物学—分子生物学] S917.4[农业科学—水产科学]