文章摘要
刘斐斐,崔晓羽,董赛赛,段胜华,汪桂玲,李家乐.三角帆蚌中WNT4基因克隆及表达分析[J].上海海洋大学学报,2020,29(6):801-810
三角帆蚌中WNT4基因克隆及表达分析
Cloning and expression analysis of WNT4 gene in the Hyriopsis cumingii
投稿时间:2019-12-30  修订日期:2020-02-07
DOI:10.12024/jsou.20191202894
中文关键词: 三角帆蚌  性别决定  WNT4  qRT-PCR  原位杂交
英文关键词: Hyriopsis cumingii  sex determination  WNT4  qRT-PCR  in situ hybridization
基金项目:国家自然科学基金(31772835);国家重点研发计划“蓝色粮仓科技创新”专项(2018YFD0901406)
作者单位E-mail
刘斐斐 上海海洋大学 农业农村部淡水水产种质资源重点实验室, 上海 201306  
崔晓羽 上海海洋大学 农业农村部淡水水产种质资源重点实验室, 上海 201306  
董赛赛 上海海洋大学 农业农村部淡水水产种质资源重点实验室, 上海 201306  
段胜华 上海海洋大学 农业农村部淡水水产种质资源重点实验室, 上海 201306  
汪桂玲 上海海洋大学 农业农村部淡水水产种质资源重点实验室, 上海 201306
上海海洋大学 上海市水产养殖工程技术研究中心, 上海 201306
上海海洋大学 水产科学国家级实验教学示范中心, 上海 201306 
glwang@shou.edu.cn 
李家乐 上海海洋大学 农业农村部淡水水产种质资源重点实验室, 上海 201306
上海海洋大学 上海市水产养殖工程技术研究中心, 上海 201306
上海海洋大学 水产科学国家级实验教学示范中心, 上海 201306 
 
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中文摘要:
      为进一步了解WNT4在三角帆蚌性别决定过程中的作用,通过RACE(Rapid-amplification of cDNA ends)技术克隆获得了三角帆蚌WNT4基因的全长cDNA,共1 560 bp,其中5'-UTR 396 bp、3'-UTR 24 bp,开放阅读框(ORF)为1 140 bp,可以编码379个氨基酸,且该序列含有WNT家族特有结构域。对WNT4的氨基酸序列进行同源性分析后,发现该基因在物种间的进化关系和结构功能具保守性。通过实时荧光定量PCR(qRT-PCR)分析发现WNT4基因在三角帆蚌雌雄多个组织(外套膜、闭壳肌、鳃、性腺、斧足和肝)中均有表达,表明其参与了多样的生物学过程,在鳃、斧足和肝中表达量较高,在性腺、闭壳肌和斧足中雌雄间有显著性差异,且雌性三角帆蚌的性腺、闭壳肌表达量显著高于雄性。原位杂交结果显示,WNT4在雌性三角帆蚌的卵母细胞上有明显的杂交信号,推测WNT4基因与性别决定相关。
英文摘要:
      In order to further understand the role of WNT4 in the sex determination process of Hyriopsis cumingii, the full-length cDNA of WNT4 gene was cloned by RACE method(Rapid-amplification of cDNA ends), which was 1 560 bp, including 5'-UTR 396 bp and 3'-UTR 24 bp. The open reading frame (ORF) was 1 140 bp, encoded a putative protein of 379 amino acids that contained a WNT family specific domain. Homology analysis of the amino acid sequence of WNT4 showed that the evolutionary relationship and structural function of the gene were conserved among species. Real-time quantitative PCR (qRT-PCR) analysis of WNT4 gene in the male and female tissues (the mantle, the adductor muscle, the gill, the gonad, the foot and the liver), indicated that it participated in diverse biological processes. It was highly expressed in gill, foot, and liver. There was significant difference between male and female in gonads, adductor muscle, and foot, and the expression of female gonads and adductor muscle was significantly higher than that of males. In situ hybridization results showed that there was obvious hybridization signal in the female oocyte cell. It was speculated that WNT4 gene was related to sex determination.
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