大菱鲆figla基因的克隆及其在卵巢分化过程中的表达分析

Cloning and expression analysis of figla during ovarian differentiation in turbot, Scophthalmus maximus

中文关键词: 大菱鲆  figla基因  初级卵母细胞  性腺分化

英文关键词: Turbot, Scophthalmus maximus  figla  primary oocyte  gonadal differentiation

中文摘要:

      Figla(factor in the germ cell line, alpha)是生殖细胞中特异性表达的转录因子之一,对于调控鱼类原始卵泡形成和卵巢发育起着关键作用。为了明确大菱鲆figla基因序列及其在卵巢早期发育过程中的功能,本研究以全雌苗种为实验对象,采用RT-PCR、cDNA末端快速扩增(RACE)克隆获得大菱鲆figla基因的全长cDNA序列,并通过半定量PCR、实时荧光定量PCR(qRT-PCR)、原位杂交(ISH)对其组织表达分布和卵巢早期发育过程中时空表达模式进行了研究。结果显示,大菱鲆figla基因cDNA全长为1006 bp,开放阅读框为609 bp(150 bp-758 bp),编码202个氨基酸,具有碱性螺旋-环-螺旋(basic Helix-Loop-Helix, bHLH)序列特征；figla在大菱鲆性腺中特异表达,卵巢中的表达水平高于精巢；在不同发育时期的卵巢发育过程中,figla mRNA于孵化后25日龄(dph)性腺中开始表达,35 dph显著增加,此后直至90 dph表达水平持续增加；原位杂交结果figla mRNA主要定位于第 Ⅰ 时相初级卵母细胞胞质内。研究结果初步提示figla基因与大菱鲆卵巢和卵母细胞分化密切相关,在促进卵泡形成和性腺分化方面起到关键作用,为深入探索大菱鲆卵巢分化和发育的分子调控机制提供参考依据。

英文摘要:

      The transcription factor gene figla (factor in the germ line, alpha), a member of the bHLH family, has been extensively documented for its pivotal role in mammalian ovarian development and primordial follicle formation. However, studies on the figla gene in teleosts have been less detailed compared to mammals. Turbot (Scophthalmus maximus), an important aquaculture species in Europe and China, exhibits sexual dimorphism in growth and body size and possesses a female heterogametic sex determination (SD) system (ZW female and ZZ male). Therefore, it is crucial for understanding the mechanism of sex determination and differentiation. To elucidate the complete cDNA sequence of the figla gene in turbot and its role during early ovarian development, this study exclusively utilized all-female individuals as experimental subjects. The full-length cDNA sequence of the figla gene was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques. Tissue expression distribution, as well as spatiotemporal expression pattern during early ovarian development, were investigated through semi-quantitative RT-PCR, real-time fluorescence quantitative PCR (qRT-PCR), and in situ hybridization (ISH). The results demonstrated that the full length of the figla gene cDNA was 1006 base pairs (bp), with an open reading frame ranging from 150 bp to 758 bp, encoding a total of 202 amino acids and featuring a conserved basic helix-loop-helix (bHLH) domain. Figla exhibited specific expression within turbot"s gonads, showing higher levels in ovaries than testes. The expression pattern of figla mRNA was detected throughout various stages of ovarian development, commencing at 25 dph and progressively increasing until reaching its peak at 90 dph. In situ hybridization experiments revealed predominant localization of figla mRNA within primary oocytes" cytoplasmic region. In conclusion, the distinct expression pattern of the figla gene in male and female turbot, its localization within primary oocytes, and its expression patterns during early ovarian development suggest a crucial role of this gene in the normal development of oocytes and ovaries. However, further research is necessary to investigate its regulatory mechanisms in oocyte development and gonadal differentiation. The study on figla gene function not only enhances our understanding of sex determination and differentiation mechanisms in turbot but also provides novel insights and methodologies for sex control and breeding applications in this species.