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潘贤辉,刘奕,周康奇,胡隐昌,郑曙明,宋红梅,牟希东,杨叶欣.2018.双须骨舌鱼卵黄蛋白原基因vtg的克隆、组织表达和原核表达分析.动物学杂志,53(4):597-614.
双须骨舌鱼卵黄蛋白原基因vtg的克隆、组织表达和原核表达分析
Molecular Cloning, Expression Pattern and Prokaryotic Expression of Vitellogenin Gene (vtg) from Osteoglossum bicirrhosum
投稿时间:2017-09-14  修订日期:2018-06-22
DOI:10.13859/j.cjz.201804011
中文关键词:  双须骨舌鱼  卵黄蛋白原  克隆  组织表达  原核表达
英文关键词:Osteoglossum bicirrhosum  Vitellogenin  Cloning  Tissue expression  Prokaryotic expression
基金项目:广州市科技计划项目(No. 201707010309),渔港建设和渔业产业发展专项(No. A201701A07),国家水产种质资源平台项目(No. 2018DKA30470),广东省自然科学基金项目(No. 2014A030310397)
作者单位E-mail
潘贤辉 西南大学淡水鱼类资源与生殖发育教育部重点实验室 重庆 400715 农业部休闲渔业重点实验室广东省现代休闲渔业工程技术研究中心中国水产科学研究院珠江水产研究所 广州 510380 15002381261@163.com 
刘奕 农业部休闲渔业重点实验室广东省现代休闲渔业工程技术研究中心中国水产科学研究院珠江水产研究所 广州 510380 liuyi@prfri.ac.cn 
周康奇 西南大学淡水鱼类资源与生殖发育教育部重点实验室 重庆 400715 农业部休闲渔业重点实验室广东省现代休闲渔业工程技术研究中心中国水产科学研究院珠江水产研究所 广州 510380 2472797247@qq.com 
胡隐昌 农业部休闲渔业重点实验室广东省现代休闲渔业工程技术研究中心中国水产科学研究院珠江水产研究所 广州 510380 huyc22@163.com 
郑曙明 农业部休闲渔业重点实验室广东省现代休闲渔业工程技术研究中心中国水产科学研究院珠江水产研究所 广州 510380 zhsm22@163.com 
宋红梅 农业部休闲渔业重点实验室广东省现代休闲渔业工程技术研究中心中国水产科学研究院珠江水产研究所 广州 510380 shm1227@126.com 
牟希东 农业部休闲渔业重点实验室广东省现代休闲渔业工程技术研究中心中国水产科学研究院珠江水产研究所 广州 510380 316530191@qq.com 
杨叶欣 农业部休闲渔业重点实验室广东省现代休闲渔业工程技术研究中心中国水产科学研究院珠江水产研究所 广州 510380 103503261@qq.com 
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中文摘要:
      为探究双须骨舌鱼(Osteoglossum bicirrhosum)卵黄蛋白原vtg基因的结构和表达特征,本研究运用RACE技术克隆双须骨舌鱼vtg全长cDNA序列。序列分析发现,双须骨舌鱼vtg的cDNA全长为5 325 bp,开放阅读框(ORF)为5 121 bp,其5′和3′非编码区(UTR)分别为46 bp和159 bp,共编码1 706个氨基酸,预测蛋白质分子量约为186.79 ku。同源性分析发现,双须骨舌鱼与同科的美丽仆骨舌鱼(Scleropages formosus)相似度达到84.78%,与鲤形目、鲈形目、鲱形目、鲽形目鱼类的相似度均高于50%。系统进化树分析结果显示,本文获得的双须骨舌鱼vtg基因与骨舌鱼目聚为一类,与鳗鲡目硬骨鱼类亲缘关系较近,与双须骨舌鱼进化地位相符。荧光定量PCR检测结果表明,在雌雄鱼性腺和肝中vtg均有表达,且在雌鱼肝中的相对表达量显著高雄鱼(P < 0.05),卵巢和精巢表达量无显著差异(P > 0.05);在雌雄鱼鳃、脾、肾、肌肉、心、头肾、脑等7个组织中均极微量表达,且无显著差异(P > 0.05)。在Ⅱ、Ⅲ、Ⅳ期卵巢中vtg相对表达量依次增加,Ⅳ期显著高于Ⅱ和Ⅲ期(P < 0.05),也显著高于精巢Ⅳ期(P < 0.05);在雌鱼肝组织中呈先增后减趋势,且Ⅱ、Ⅲ、Ⅳ期之间无显著差异(P > 0.05),但每个时期都显著高于雄性(P < 0.05)。成功构建了原核表达载体pEASY-BluntE2-vtg,并转入Transetta(DE3)表达感受态细胞中成功诱导出融合蛋白。Western-blotting检测显示,融合蛋白可被抗His标签特异性识别。综上表明,vtg表达水平高低与性别相关,并与性腺发育程度有关。此外,本研究结果也为后续深入研究卵黄蛋白原的生理功能打下基础。
英文摘要:
      In order to investigate the structure and expression of vtg encoding vitellogenin (VTG) in Osteoglossum bicirrhosum, full-length cDNA sequence of vtg was cloned by the rapid-amplification of cDNA ends (RACE). Sequence analysis found that the full length of vtg was 5 325 bp, including 5 121 bp of the open reading frame (ORF), 46 bp of the 5′ untranslated region (UTR) and 159 bp of 3′ UTR. The ORF contained a total of 1 706 encoded amino acids with a predicted molecular mass of 186.79 ku. The results of domain analysis showed that there were 4 conserved domains in ORF, lipoprotein amino terminal region (vitellogenin-N), von Willebrand factor type D domain (vWD), domain of unknown function (DUF) 1943 and DUF 1944 (Fig. 2). The results of homologous analysis showed that the similarity comparing with Scleropages formosus was the highest of 84.78%, and higher than 50% comparing with Cypriniformes, Perciformes, Clupeiformes, Heterosomata. Phylogenetic analysis showed that vtg of O. bicirrhosum came to be a cluster with Osteoglossiformes, and was closely related with Anguilliformes, which was consistent with the evolutionary position of O. bicirrhosum (Fig. 5). The expression pattern of vtg was evaluated using real-time fluorescent quantitative PCR, and the results showed that the expression of vtg was the highest in the liver of female, significantly higher than that in males (P < 0.05). There was no significant difference in its expression between ovary and testis (P > 0.05). The expression quantity of vtg in gills, spleen, kidney, muscle, heart, head-kidney or brain of males and females was all very low (P > 0.05) (Fig. 6). The relative expression of vtg in the ovary at stage Ⅱ, Ⅲ and Ⅳ was increased successively, and its expression at the stage Ⅳ was significantly higher than that at stage Ⅱ and Ⅲ (P < 0.05), and was significantly higher than that of stage Ⅳ testis. In the liver of female vtg expression was increased at first and then decreased, and no significant difference among the 3 stages (P > 0.05) was found, but vtg expression at each stage was significantly higher than that of males (P < 0.05) (Fig. 7). We successfully constructed the prokaryotic expression vector, and transferred it to Transetta (DE3) expression competent cells to induce expression the fusion protein (Fig. 8, 9). Western-blotting showed that the fusion protein could be specifically recognized by anti-His tag (Fig. 10). In conclusion, the expression level of vtg is related to sex, and it is also related to gonadal development stage. In addition, this study has laid a foundation to further investigating the physiological function of VTG.
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