黄河鲤nanog基因克隆及其表达特征研究

Cloning and expression characteristics of nanog gene in the Yellow River carp

转录因子Nanog在干细胞多能性维持、胚胎早期发育等方面具有重要作用.利用PCR技术克隆黄河鲤nanog基因(Ccnanog),通过实时荧光定量PCR或半定量PCR分别对其在黄河鲤不同胚胎发育时期以及雌、雄成体不同组织中的表达特征进行了分析,并利用荧光原位杂交和免疫组织化学方法进一步检测了该基因在黄河鲤卵巢和精巢中的亚细胞定位.结果显示,黄河鲤nanog基因ORF为1158 bp,编码385个氨基酸残基;Nanog蛋白包含保守的HOX结构域,与斑马鱼的序列相似性最高.在胚胎中,Ccnanog在未受精卵、早期胚胎的Ⅰ细胞期至原肠胚期高表达,在神经胚期表达量显著降低,之后在眼囊期至出膜期基本检测不出;在组织中,Ccnanog短片段在以性腺为首的多个组织中均有不同程度的表达,Ccnanog长片段仅在雌鱼卵巢和皮肤中表达,其中在雌鱼卵巢中的表达量最高,其组织表达具有一定的性别二态性;在性腺中进一步检测到,Ccnanog主要分布在卵巢的卵原细胞、Ⅰ期、Ⅱ期和Ⅲ期初级卵母细胞的细胞质中,以及精巢的精原细胞、精母细胞和精子细胞中,且随着生殖细胞的发育其表达量逐渐下降.以上结果表明Ccnanog可能参与了黄河鲤早期胚胎发育过程与生殖细胞的发育,为后续功能研究提供了参考.

The transcription factor Nanog plays an important role in maintenance of pluripotency of stem cells and early embryonic development.In this study,the nanog gene of the Yellow River carp(Ccnanog)was cloned by PCR technology.The expression characteristics of Ccnanog gene in different embryonic development stages and in different tissues of female and male adults of the Yellow River carp were analyzed by real-time fluorescence quantitative PCR or semi-quantitative PCR.The subcellular localization of Ccnanog gene in ovary and testis of the Yellow River carp were further detected by fluorescence in situ hybridization and immunohistochemistry.The results showed that the ORF of the nanog gene of the Yellow River carp was 1158 bp,encoding 385 amino acid residues.Nanog protein contains a conserved HOX domain,which has the highest sequence similarity with zebrafish.In the embryo,Ccnanog was highly expressed in the unfertilized eggs,from the 1-cell stage to the gastrula stage,and the expression level significantly decreased in the neurula stage,then was basically undetectable from the optic capsule stage to the hatching stage.In tissues,short segments of Ccnanog are expressed to varying degrees in multiple tissues headed by the gonads,while long segments of Ccnanog are only expressed in the ovary and skin of female fish,with the highest expression level in the ovary,and their tissue expression has a certain gender dimorphism.It was further detected in the gonads that Ccnanog was mainly distributed in the oogonia,cytoplasm of the phase I,II and III primary oocytes of the ovary,as well as in the spermatogonia,spermatocytes and sperm cells of the testis,and their expression gradually decreased with the development of the oocytes or spermatocytes.The above results suggest that Ccnanog may participate in the early embryos and germ cells development of the Yellow River carp,providing a reference for the follow-up study of its function.