生殖细胞的鉴定和表达对于研究鱼类性别相关机制具有重要意义。Vasa基因,编码ATP依赖性RNA解旋酶,被认为是生殖细胞的分子标记,在生殖细胞发育中起着至关重要的作用。asotusasotus,是中国重要的淡水经济鱼类,显示出明显的性别二态性,女性的生长速度比男性快。然而,这些性别差异背后的分子机制,特别是涉及这种鱼的vasa基因的分子机制仍然知之甚少。在这项工作中,通过RT-PCR和cDNA末端快速扩增(RACE)获得了asotus的vasa基因序列。并使用qRT-PCR和原位杂交方法分析其在胚胎和组织中的表达。还对幼虫鱼进行来曲唑(LT)处理,以研究其对基因的影响。结果表明,Savasa的开放阅读框(ORF)为1989bp,编码662个氨基酸。SaVasa蛋白包含DEAD-box蛋白家族特有的10个保守结构域,与丝虫的序列同一性最高,为95.92%。在胚胎中,Savasa在早期胚胎中从双细胞期到囊胚期高度表达,从胃动期到心跳期呈逐渐下降的趋势。此外,Savasa最初是在双细胞阶段的卵裂沟结束时检测到的,后来随着胚胎发育浓缩成四个对称的细胞簇。在胃肠病阶段,Savasa阳性细胞增加并开始向胚胎的背侧迁移。在组织中,Savasa主要在卵巢中表达,在其他检测到的组织中几乎没有表达或表达较低。此外,Savasa在卵巢I-V期卵母细胞中表达,以及睾丸中的精原细胞和精母细胞,暗示生殖细胞的特定表达模式。此外,在鱼的关键性腺分化期间,LT以浓度依赖性方式显着上调Savasa的表达。值得注意的是,LT治疗后120dph,高浓度组的睾丸和卵巢中Savasa表达最低。总的来说,来自基因结构的发现,蛋白质序列,系统发育分析,RNA表达模式,对LT的反应表明Savasa是母系遗传的,具有保守的特征,作为S.asotus生殖细胞的潜在标记基因,并可能参与LT诱导的鱼类早期胚胎发育和性腺发育过程。这将为进一步研究生殖细胞标记物的应用以及沙门菌性别差异的分子机制奠定基础。
The identification and expression of germ cells are important for studying sex-related mechanisms in fish. The vasa gene, encoding an ATP-dependent RNA helicase, is recognized as a molecular marker of germ cells and plays a crucial role in germ cell development. Silurus asotus, an important freshwater economic fish species in China, shows significant sex dimorphism with the female growing faster than the male. However, the molecular mechanisms underlying these sex differences especially involving in the vasa gene in this fish remain poorly understood. In this work, the vasa gene sequence of S. asotus (named as Savasa) was obtained through RT-PCR and rapid amplification of cDNA end (RACE), and its expression in embryos and tissues was analyzed using qRT-PCR and an in situ hybridization method. Letrozole (LT) treatment on the larvae fish was also conducted to investigate its influence on the gene. The results revealed that the open reading frame (ORF) of Savasa was 1989 bp, encoding 662 amino acids. The SaVasa protein contains 10 conserved domains unique to the DEAD-box protein family, showing the highest sequence identity of 95.92% with that of Silurus meridionalis. In embryos, Savasa is highly expressed from the two-cell stage to the blastula stage in early embryos, with a gradually decreasing trend from the gastrula stage to the heart-beating stage. Furthermore, Savasa was initially detected at the end of the cleavage furrow during the two-cell stage, later condensing into four symmetrical cell clusters with embryonic development. At the gastrula stage, Savasa-positive cells increased and began to migrate towards the dorsal side of the embryo. In tissues, Savasa is predominantly expressed in the ovaries, with almost no or lower expression in other detected tissues. Moreover, Savasa was expressed in phase I-V oocytes in the ovaries, as well as in spermatogonia and spermatocytes in the testis, implying a specific expression pattern of germ cells. In addition, LT significantly upregulated the expression of Savasa in a concentration-dependent manner during the key gonadal differentiation period of the fish. Notably, at 120 dph after LT treatment, Savasa expression was the lowest in the testis and ovary of the high concentration group. Collectively, findings from gene structure, protein sequence, phylogenetic analysis, RNA expression patterns, and response to LT suggest that Savasa is maternally inherited with conserved features, serving as a potential marker gene for germ cells in S.asotus, and might participate in LT-induced early embryonic development and gonadal development processes of the fish. This would provide a basis for further research on the application of germ cell markers and the molecular mechanisms of sex differences in S. asotus.