【摘要】 在鱼类的早期胚胎发育中生殖细胞便与体细胞分离,形成所谓的原始生殖细胞(primordial germ cells,PGCs)。母源性基因Dead end(dnd)在胚胎发育中特异地表达于原始生殖细胞,编码一种进化上保守的RNA结合蛋白,对生殖细胞发育具有重要作用。本研究通过PCR获得大黄鱼dnd基因(Lcdnd)的部分c DNA序列,结合实时定量PCR、整胚原位杂交等技术,研究Lcdnd在各组织和胚胎发育中的表达。实时定量PCR结果显示:dnd在性腺中特异表达,且其在卵巢中的表达量高于精巢;检测的各期胚胎中都有dnd的表达,随着胚胎发育其表达量呈下降趋势。整胚原位杂交结果显示:Lcdnd在早期卵裂阶段主要分布于分裂沟,到囊胚期至原肠早期则开始定位于细胞内,原肠早期时定位在胚环中的中内胚层,且阳性信号增多,暗示此时原始生殖细胞的形成。接着原始生殖细胞沿胚环向胚体形成处(胚盾)迁移;随着胚胎的发育,发现胚体两侧的阳性信号增多;到体节发生期,阳性信号细胞分布于胚体两侧的侧板中胚层,随着体节的发生这些细胞沿背腹轴向腹部运动;发育到孵出期时,到达了发育中的原始生殖嵴。本研究首次使用dnd作为较为可靠的大黄鱼生殖细胞标记,揭示了大黄鱼原始生殖细胞的起源与迁移,为大黄鱼生殖细胞发生发育及养殖大黄鱼的育性控制研究奠定一定的基础。更多还原

【Abstract】 In many fishes,germ cells originate from primordial germ cells( PGCs) and are segregated from somatic cell lineage during embryogenesis. Dead end( dnd),a maternal gene,is exclusively expressed in PGCs and encodes an evolutionary conserved RNA-binding protein,playing a critical role in germline cell development during embryogenesis. In this study,according to the nucleotide sequences of dnd from large yellowcroaker( Larimichthys crocea) in NCBI database,partial c DNA sequence was cloned by PCR and denoted as Lcdnd. M eanwhile,we addressed the expression analysis of Lcdnd by means of real-time quantitative PCR( qRT-PCR) and whole mount in situ hybridization( WISH). Results showed that Lcdnd specifically expressed in gonad and expression level in ovary were significantly higher than those in testis.Results showed that the expressions of Lcdnd were detected in all stages examined during embryogenesis.Lcdnd tended to gradually decrease as the embryo developed. Furthermore,results of WISH showed that Lcdnd transcripts were localized to the cleavage furrowduring early cleavage stages. As the embryo developed to blastula stage and early-gastrula stage,the increasing positive signals started to locate at a small number of cells that means the specification of future primordial germ cells. M oreover,the dnd-positive cells were mainly distributed in the mesentoderm of germ ring region at early-gastrula stage. During subsequent stages,putative increasing PGCs migrated to the embryonic shield region along the germ ring and then the dnd-positive cells increased at both sides of the trunk. At somitogenesis stage,the dnd-positive cells were distributed as two lines located in lateral mesoderm. Subsequently,those cells migrated toward the abdomen of the trunk along the dorsal-ventral axis and then clustered in the putative primitive genital ridge. In short,we successfully used Lcdnd gene as a germ cell marker for the first time to elucidate the origin and migration of PGCs of large yellowcroaker,and laid the foundation for further studies on germ cell specification and development and the fertility control.更多还原