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刘晓莉,唐舟凯,张飘逸,朱新平,储张杰,李伟,吴栩灵,徐红艳.2019.中华鳖boule基因在生殖细胞中的表达分析.动物学杂志,54(1):66-75.
中华鳖boule基因在生殖细胞中的表达分析
The Expression Profile of boule Gene in Germ Cells of the Chinese Soft-shell Turtle (Pelodiscus sinensis)
投稿时间:2018-08-09  修订日期:2018-12-11
DOI:10.13859/j.cjz.201901009
中文关键词:  中华鳖  boule  生殖细胞  精子发生  卵子发生
英文关键词:Chinese Soft-shell Turtle, Pelodiscus sinensis  boule  Germ cell  Spermatogenesis  Oogenesis
基金项目:广东省科技计划项目(No. 2016A050502029),社会公益研究项目(No. 9180238)
作者单位E-mail
刘晓莉 中国水产科学研究院珠江水产研究所 农业部热带亚热带水产资源利用与养殖重点实验室 广州 510380 liu_xiaoli1988@126.com 
唐舟凯 中国水产科学研究院珠江水产研究所 农业部热带亚热带水产资源利用与养殖重点实验室 广州 510380浙江海洋大学 水产学院 浙江舟山 316000 1252950615@qq.com 
张飘逸 中国水产科学研究院珠江水产研究所 农业部热带亚热带水产资源利用与养殖重点实验室 广州 510380 zhangpiaoyi00000@126.com 
朱新平 中国水产科学研究院珠江水产研究所 农业部热带亚热带水产资源利用与养殖重点实验室 广州 510380 zhuxinping_1964@163.com 
储张杰 浙江海洋大学 水产学院 浙江舟山 316000 czj0501@sina.com 
李伟 中国水产科学研究院珠江水产研究所 农业部热带亚热带水产资源利用与养殖重点实验室 广州 510380 664061430@qq.com 
吴栩灵 中国水产科学研究院珠江水产研究所 农业部热带亚热带水产资源利用与养殖重点实验室 广州 510380 564155192@qq.com 
徐红艳 中国水产科学研究院珠江水产研究所 农业部热带亚热带水产资源利用与养殖重点实验室 广州 510380 xuhyzqh@163.com 
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中文摘要:
      boule基因为DAZ基因家族成员之一,是动物生殖细胞特异表达基因。在哺乳动物中,boule基因的缺失会引起精子生成障碍而导致雄性不育。在脊椎动物秀丽线虫(Caenorhabditis elegans)中,boule基因同源物的缺失会引起其卵子发生障碍而导致雌性不育。龟鳖动物是最古老的爬行类,是从羊膜卵到羊膜卵动物飞跃的过渡物种。相比于哺乳类及一些脊椎动物,目前关于龟鳖动物生殖细胞发育模式的研究还非常有限。因此,本文以中华鳖(Pelodiscus sinensis)为研究对象,以期揭示boule基因对龟鳖动物生殖细胞发育分化的调控作用。首先,利用特异引物克隆获得中华鳖boule基因的cDNA序列,共1 005 bp,其中,3′端非编码区57 bp,开放阅读框948 bp,共编码315个氨基酸。氨基酸序列多重比对分析显示,中华鳖与绿海龟(Chelonia mydas)同源性最高,达92%,与小鼠(Mus musculus)的同源性达83%,与果蝇(Drosophila melanogaster)的同源性达53%,与青鳉(Oryzias latipes)的同源性达42%。反转录实时定量PCR(RT-qPCR)分析结果显示,中华鳖boule mRNA主要在性腺组织精巢和卵巢中表达,而在其他体细胞组织中几乎检测不到表达。原位杂交结果显示,中华鳖boule mRNA在两性生殖细胞中特异表达,且在不同分化时期的生殖细胞中呈动态表达。在精巢中,boule mRNA在初级精母细胞中表达最强,在精原细胞和次级精母细胞中表达较弱,在精子细胞和精子中难以检测到表达信号;在卵巢中,boule mRNA在初级卵母细胞中表达信号最强且信号在初级卵母细胞胞质中均匀分布,生殖细胞发育进入卵母细胞生长期后,信号开始聚集在核周胞质,随着卵母细胞的成熟,信号逐渐变弱。本研究结果表明,boule基因可能在中华鳖两性生殖细胞的减数分裂过程中均具有重要的调控作用。
英文摘要:
      Boule gene, one of the DAZ (Deleted in Azoospermia) gene family members, is specifically expressed in animal germ cells. In mammals, the boule null caused spermatogenic failure, leading to male infertility, while the loss of boule in invertebrate, such as Caenorhabditis elegans resulted in female sterility due to a failure of oogenesis. Turtles are considered as a link between the high and low vertebrates, however, the molecular mechanisms behind germ cell development in reptile including turtles remain largely unclear. In this study, we used the Chinese Soft-shell Turtle (Pelodiscus sinensis) as a model to reveal the mechanisms under germ cell differentiation in turtles. Firstly, we cloned a turtle boule cDNA fragment of 1 005 bp, containing a 3′ untranslated region (UTR) of 57 bp and an open reading frame (ORF) of 948 bp, encoding 315 amino acid residues (Fig. 1). The predicted turtle Boule is maximally 92% identical to that of Chelonia mydas, 83% to that of mouse, 53% to that of Drosophila melanogaster and 42% to that of Oryzias latipes (Fig. 2). The reverse transcription quantitative real-time PCR (RT-qPCR) showed that boule transcript of P. sinensis was abundantly expressed in adult ovary and testis but barely in all the somatic tissues examined in this study (Fig. 4). Chemical in situ hybridization revealed that the boule mRNA of P. sinensis was exclusively expressed in germ cells but absent in somatic cells, and displayed dynamic expression patterns in germ cells during gametogenesis (Fig. 5 & 6). In testis, the boule mRNA signals were strongly dispersed in cytoplasm of primary spermatocytes and weakly in spermatogonia and secondary spermatocyte, hardly detected in spermatids and spermatozoa (Fig. 5). In ovary, the boule mRNA signals were strongly dispersed in cytoplasm of oocytes at early stages. Subsequently, the signals were concentrated in the perinuclear area of growing oocytes and weakened gradually with the growth of oocytes (Fig. 6). Therefore, our findings indicated that boule gene might play an important role in germ cell meiosis in Pelodiscus sinensis, which shed new insight into understanding the functions of boule gene during germ cell differentiation in vertebrates.
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