National Natural Science Foundation of China (31873035); Fundamental Research Funds for the Central Universities (SWU020014)
为了研究龟鳖类生殖细胞发育分化机制，实验通过RACE技术克隆获得了中华鳖dead end (dnd) 基因全长cDNA，RT-PCR分析了其在不同组织的转录表达情况，并通过原位杂交技术检测了dnd mRNA在中华鳖雌雄性腺配子发生过程中的表达变化。结果显示，中华鳖dnd cDNA 全长1 251 bp (GenBank登录号为OL757532)，包括234 bp的3' 端非翻译区和1 017 bp开放读码框，开放读码框编码338个氨基酸。氨基酸序列比对和系统进化树分析结果表明，中华鳖Dnd蛋白与其他物种同源蛋白一样，均具有6个保守的结构域，其中高度保守的RNA识别结构域是主要的功能结构域。中华鳖Dnd与绿海龟Dnd氨基酸序列一致性最高，亲缘关系最近。RT-PCR结果显示，Psdnd特异性地在中华鳖性腺组织中表达，且在卵巢中的表达明显高于精巢。原位杂交分析结果显示，Psdnd mRNA在生殖细胞中特异表达。在卵巢中，Psdnd mRNA主要在Ⅱ期初级卵母细胞的细胞质中表达，随着卵母细胞的增大，在Ⅳ期及以后的卵母细胞中检测不到其表达。在精巢中，Psdnd mRNA信号在初级精母细胞中表达最强，在次级精母细胞和精原细胞中表达水平次之，在精子细胞中未检测到信号。研究表明，dnd基因可能在调控中华鳖雌雄生殖细胞的发育中起重要作用。
Dead end (dnd) is a germ cell-specific molecular marker in most vertebrates, belonging to RNA-binding protein family. It is essential for the maintenance of the survival and migration of primordial germ cells (PGCs). In many species, dnd deficiency could block the migration of PGCs, and then they transdifferentiate into other cell types, resulting in complete loss of germ cells and sterility. Thus, dnd is essential for the survival and development of vertebrate germ cells. Dnd is originally identified in Danio rerio, proved that deletion of Dnd leads to abnormal polarization and migration of PGCs and loss of germ cells. Thereafter, dnd1 is characterized in Mus musculus and Dnd1-β is specifically expressed in adult testis. However, chicken Dnd homologue (CDH) is expressed in both gonads. In Xenopus laevis, dnd is only expressed in ovary. In teleosts, dnd has been identified as a PGCs marker and is specifically expressed in gonads that the expression level is higher in ovary than testis. Therefore, dnd shows distinct expression patterns among species. Although dnd plays a conserved role in maintaining the survival and migration of PGCs from teleosts to mammals, its expression pattern in gametogenesis varies with species. Thus, it is of great significance to identify the sequence characteristics, gene expression and distribution of dnd in turtles, to lay the foundation for studying on the origin and formation of PGCs, and gametogenesis of turtles. Pelodiscus sinensis is one of the oldest tetrapods and has unique scientific research value in study of animal morphological and reproductive evolution. Additionally, because of its high medicinal and economic values, P. sinensis occupies an important position in freshwater aquaculture in China. Here, in order to study the development and differentiation mechanism of turtles’ germ cells, the full-length cDNA of dnd was obtained by RACE form P. sinensis. The total length of P. sinensis dnd (Psdnd) cDNA was 1 251 bp (GenBank ID OL757532), including 254 bp 3' untranslated region and 1 017 bp open reading frame encoding 338 amino acids. Multiple alignment and phylogenetic tree analysis showed that Psdnd had 6 conserved domains, the same as homologous proteins of other species had, and the highly conserved RNA recognition domain was the main functional region. Besides, Psdnd had the highest sequence identity with and was the closest relative to Chelonia mydas dnd. RT-PCR showed that Psdnd was specifically expressed in gonads and the mRNA level was higher in ovary than testis. Chemical in situ hybridization showed that Psdnd mRNA was specifically expressed in germ cells. Particularly, Psdnd mRNA was mainly expressed in the cytoplasm of stage Ⅱ primary oocytes, and not expressed in oocytes stage Ⅳ onwards. Moreover, Psdnd mRNA signals were strongly detected in primary spermatocytes, followed by secondary spermatocytes and spermatogonia, and barely in spermatids. Our study indicates that dnd may play a key role in regulating the development of male and female germ cells in P. sinensis. This study lays the basis for further study of turtles’ germ cells development and differentiation mechanism.