文章摘要
崔晓雨,朱冬发,汤 洁,谢 熙,邱锡尔.三疣梭子蟹EcR基因的克隆及表达分析[J].水产学报,2013,37(11):1645~1654
三疣梭子蟹EcR基因的克隆及表达分析
Cloning and expression analysis of ecdysteroid receptor (EcR)in Portunus trituberculatus
投稿时间:2013-08-09  修订日期:2013-09-22
DOI:DOI:10.3724/SP.J.1231.2013.38847
中文关键词: 三疣梭子蟹  蜕皮激素受体  蜕皮周期  二次卵巢发育
英文关键词: Portunus trituberculatus  ecdysteroid receptor(EcR)  molting stage  second ovarian development
基金项目:国家自然科学基金项目(41376152,40976098);浙江省自然科学基金项目(LY13C190006);宁波市自然科学基金项目(2012A610137)
作者单位
崔晓雨 宁波大学海洋学院 
朱冬发 宁波大学海洋学院 
汤 洁 宁波大学海洋学院 
谢 熙 宁波大学海洋学院 
邱锡尔 宁波大学海洋学院 
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中文摘要:
      为研究蜕皮激素受体(EcR)在甲壳动物生长和生殖过程中的作用,采用反转录PCR(RT-PCR)和cDNA 末端快速扩增(RACE)技术,克隆了三疣梭子蟹蜕皮激素受体 (PtEcR)基因全长cDNA序列(GenBank登录号:KC354381),运用实时荧光定量PCR(qRT-PCR)方法,分析该基因在不同组织、蜕皮时期和二次卵巢发育阶段的表达特 征。结果表明,PtEcR cDNA全长2 231 bp,包含1 269 bp ORF,编码422个氨基酸;推导的PtEcR氨基酸序列与已公布的其他甲壳动物EcR进行比对,发现一致性 达67%~97%;系统进化树分析PtEcR与其它甲壳动物EcR聚为一支,而昆虫EcR聚为另一支。PtEcR在检测的三疣梭子蟹10个组织中均有表达,其中在Y-器(YO)表达量最高。在蜕皮周期中,自蜕皮后期至蜕皮前期D2亚期PtEcR在YO中的表达量一直较低;至D3亚期和D4亚期,PtEcR表达量显著升高,这与血 淋巴中20-羟基蜕皮酮(20E)浓度在D3/D4亚期显著升高相协同,表明PtEcR在三疣梭子蟹蜕皮过程中起着重要的作用。二次卵巢发育阶段,PtEcR在YO和肝胰 腺(Hp)中的表达量自Ⅱ期逐渐升高至Ⅳ期达到最高;卵巢(Ov)中,则在Ⅰ、Ⅲ期较低,Ⅱ、Ⅳ期较高,表明PtEcR可能参与卵巢发育和卵黄发生。
英文摘要:
      To study the regulatory role of ecdysteroid receptor(EcR)in molting and ovarian development of crustaceans,the full-length EcR cDNA of Portunus trituberculatus(PtEcR)was cloned by using reverse transcript PCR(RT-PCR)and rapid amplification of cDNA ends(RACE).The full-length of PtEcR(GenBank accession number:KC354381)was 2 231 bp,included a 1 269 bp ORF which encoded 422 amino acid residues.The alignment of amino acid sequence of PtEcR and that of other crustaceans showed that their identities were 67% to 97%.Phylogenetic analysis of EcR showed PtEcR was clustered in crustaceans EcRs and separated from insect EcRs.Quantitative real-time PCR(qRT-PCR)was used to quantify the relative expression level of PtEcR in different tissues,molting process and the second ovarian development in P.trituberculatus .PtEcR was expressed in various tissues and highest in the Y-organ(YO).During the molting process,the expression levels of PtEcR in YO remained low from postmolt period to substages of D2 premolt period,then significantly increased at substages D3 and D4.The changes in the expression levels of PtEcR were consistent with those in the levels of hemolymphatic 20-hydroxyecdysone,which demonstrated that PtEcR played an important role in molting regulation of P.trituberculatus.During the second ovarian development,the expression levels of PtEcR in YO and hepatopancreas gradually increased to the maximum from stage Ⅱ to Ⅳ;The expression levels of PtEcR in ovary at stages Ⅱ and Ⅳ were significantly higher than those at stage Ⅰ and Ⅲ.The results indicated that PtEcR may play an important role during ovarian development in P.trituberculatus.
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