中华鳖vasa基因克隆及在卵母细胞中的表达分析

发布时间：2018-03-25 18:38

本文选题：vasa　切入点：中华鳖　出处：《水生生物学报》2017年02期

【摘要】：研究利用中华鳖为研究模型进行爬行类生殖细胞发育分化成熟等生物学研究,克隆了中华鳖vasa基因的c DNA序列,全长3865 bp,包括5′端非编码区90 bp,3′端非编码区1699 bp,开放阅读框长2076 bp,共编码691个氨基酸。中华鳖Vasa氨基酸序列包含DEAD-box家族蛋白8个保守保守功能域,在N末端有4个RGG重复序列和2个GG富集区,与小鼠Vasa蛋白的同源性较高(72%)。荧光定量PCR的结果表明,中华鳖vasa m RNA主要精巢和卵巢中表达,其他体组织中均难检测到表达。卵巢冰冻切片原位杂交结果显示:中华鳖vasa m RNA在生殖细胞中特异表达;在卵子发生过程中的不同发育期卵母细胞中呈现动态的变化。即vasa m RNA在初级卵母细胞及生长期卵母细胞中表达最强,且均匀分布在细胞质中,随着卵母细胞的逐渐增大,信号逐渐减弱,直至在成熟的卵母细胞中几乎检测不到表达信号,说明vasa可能在中华鳖早期卵母细胞发育中起重要作用。同时,vasa基因可作为中华鳖生殖细胞分子标记物,根据其m RNA的表达水平来鉴别不同发育时期的卵母细胞。研究结果为进一步开展中华鳖胚胎生殖细胞发育及配子生成,特别是研究中华鳖,乃至爬行类原始生殖细胞(Primordial Germ Cells,PGCs)的起源、迁移、分化等研究奠定了基础。
[Abstract]:The c DNA sequence of vasa gene of Trionyx sinensis (Trionyx sinensis) was cloned by using the model of development, differentiation and maturation of reptile germ cells. The total length of 3865 BP, including the 5 '-terminal non-coding region of 90bp and the 3'-terminal region of 1699 BP, and the length of the open reading frame of 2076 BP, encode 691 amino acids. The Vasa amino acid sequence of Trionyx sinensis contains 8 conserved functional domains of the DEAD-box family protein. There were 4 RGG repeats and 2 GG rich regions at the N-terminal, which had high homology with mouse Vasa protein. The results of fluorescence quantitative PCR showed that vasa m RNA was mainly expressed in spermary and ovary of Trionyx sinensis. The results of in situ hybridization of frozen sections of ovary showed that vasa m RNA was expressed specifically in germ cells of Trionyx sinensis. The expression of vasa m RNA was the strongest in the primary oocytes and oocytes in the growing stage, and distributed uniformly in the cytoplasm, and increased with the increase of the oocytes. The signal was gradually weakened until almost no expression signal was detected in mature oocytes, indicating that vasa may play an important role in the early oocyte development of Trionyx sinensis, and Vasa gene could be used as a molecular marker of germ cells of Trionyx sinensis. According to the expression level of m RNA, the oocytes of different developmental stages were identified. The results showed that the embryonic germ cell development and gametophysiogenesis of Trionyx sinensis were further developed, especially in Trionyx sinensis. The origin, migration and differentiation of primordial Germ CellsCss of reptile primordial germ cells have laid a foundation for the study of the origin, migration and differentiation of primordial germ cells.
【作者单位】：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;上海海洋大学水产与生命学院;
【基金】：广东省科技计划项目-国际科技合作领域(2016A050502029);广东省科技计划项目(2014A020208037) 引进国际先进农业科学技术‘948’项目(2015-Z019)资助~~
【分类号】：S917.4

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