脊尾白虾EcR基因的克隆及其在卵巢和胚胎发育过程中的表达分析
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河南师范大学水产学院,中国水产科学研究院南海水产研究所,中国水产科学研究院南海水产研究所,中国水产科学研究院黄海水产研究所,中国水产科学研究院黄海水产研究所,中国水产科学研究院黄海水产研究所,中国水产科学研究院黄海水产研究所,河南师范大学水产学院

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Q785;S917

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国家虾产业技术体系专项(CARS-47);公益性行业(农业)科研专项(201103034)


Molecular cloning of ecdysteroid receptor and its expression during the ovarian development and embryogenesis of Exopalaemon carinicauda
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College of Fisheries,Henan Normal University,Xinxiang,South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao,College of Fisheries,Henan Normal University,Xinxiang

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    摘要:

    为了解蜕皮激素受体(EcR)在脊尾白虾卵巢和胚胎发育中的作用,采用同源克隆和RACE技术,克隆了脊尾白虾EcR基因全长cDNA序列(GenBank登录号:KC506600),用实时荧光定量PCR方法分析了EcR基因在雌虾不同组织、卵巢以及胚胎发育不同时期的表达特征。结果显示,脊尾白虾EcR基因全长2 638 bp,开放阅读框1 713 bp,编码570个氨基酸,脊尾白虾EcR在系统进化上与行抱卵繁殖的真虾类和蟹类亲缘关系较近,而与对虾类较远。脊尾白虾EcR基因在各组织中均有表达,但主要在肝胰腺内表达。在脊尾白虾繁殖期内,肝胰腺内EcR的表达量始终高于卵巢,表达量峰值出现在卵巢成熟时期,而卵巢内EcR的表达量在卵巢成熟前期达到最大,两种组织中EcR表达量与卵黄蛋白原(Vg)质量浓度变化趋势相一致。随着胚胎发育,EcR表达量呈先上升后下降趋势,在原肠期达到最大,前溞状幼体期和溞状幼体Ⅰ期的表达量虽有下降,但仍保持较高水平。结果表明,脊尾白虾EcR基因系统进化方向与虾蟹类不同繁殖方式相吻合,其mRNA主要在肝胰腺内表达;卵巢发育过程中,EcR基因参与了肝胰腺和卵巢中Vg合成,其在肝胰腺中表达量变化与性腺发育指数变化趋势一致,对卵巢成熟有重要作用;在胚胎发育过程中,EcR基因参与了胚胎细胞分化及器官形成。

    Abstract:

    The ridgetail white prawn,Exopalaemon carinicauda supports a large yield of polyculture ponds in eastern China.However,the artificial breeding technique for E.carinicauda is not quite advanced,which limits the further development of the E.carinicauda culture,and there have been few studies on the mechanism of gonadal development and embryonic development.Recent studies have shown that ecdysteroid receptor(EcR)is not only involved in molting and growth of Arthropod but also plays important roles in ovarian development and embryogenesis.To study the regulatory role of EcR in ovarian development and embryogenesis of E.carinicauda,the full-length EcR cDNA of E.carinicauda(EcEcR)was cloned by the methods of degenerated oligonucleotide primers and rapid amplification of the cDNA ends(RACE),and quantitative real-time PCR was used to quantify the relative expression level of EcEcR in different tissues,ovarian development and embryogenesis of E.carinicauda.The results showed that the full-length EcEcR(GenBank accession number:KC506600)was 2 638 bp,containing an open reading frame(ORF)of 1 713 bp encoding a 570 amino acid polypeptide.Multi-sequence alignment analysis showed that EcEcR shared the high identities with Crangon crangon,Macrobrachium nipponens,Homarus americanus,however,it shared lower identity with EcRs from Marsupenaeus japonicas and Litopenaeus vannamei.EcEcR was expressed in various tissues and the highest in the heptopancreas.The EcEcR mRNA levels in ovary and heptopancreas of E.carinicauda reached a maximum at the stage Ⅲ and stage Ⅳ,respectively,and the EcEcR expression levels were higher in heptopancreas than in ovary during the ovarian development.However,the EcEcR mRNA levels in ovary at stages IV and V were significantly higher than those at stages Ⅰ and Ⅱ.Another very important finding was that significantly positive correlations existed between the EcEcR expression level and total vitellogenin content.During embryogenesis,the levels of EcEcR expression were higher at stages of gastula,protozoea and metazoea than those at other stages.The results indicated that the relationships displayed in the phylogenic tree of EcRs may correspond to their breeding practice,and EcEcR mRNA was expressed highly in hepatopancreas.EcEcR was involved in vitellogenin synthesis during ovarian development of E.carinicauda, and the EcEcR expression level in hepatopancreas was consistent with the gonadosomatic index.EcEcR played an important role in the ovarian maturation,and may play key roles in cell differentiation and organogenesis during embryo development.

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梁俊平,王芸,段亚飞,葛倩倩,李吉涛,李健,刘萍,聂国兴.脊尾白虾EcR基因的克隆及其在卵巢和胚胎发育过程中的表达分析[J].水产学报,2015,39(7):942~952

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  • 收稿日期:2014-10-23
  • 最后修改日期:2015-04-29
  • 录用日期:2015-07-01
  • 在线发布日期: 2015-07-20
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