中华蛸vasa基因的克隆及表达分析

Cloning and Expression of the vasa Gene in the Octopus sinensis

关键词:  中华蛸  vasa  基因克隆  表达分析

Key words:  Octopus sinensis  vasa  Gene cloning  Expression analysis

摘要:

vasa基因编码的蛋白是DEAD-box (Asp-Glu-Ala-Asp)蛋白家族成员，对真核生物生殖细胞的形成具有关键作用。本研究使用cDNA末端快速扩增技术(RACE)克隆了中华蛸(Octopus sinensis) vasa基因全长，共2438 bp，其中，开放阅读框长2067 bp，编码688个氨基酸，5′-UTR长128 bp，3′-UTR长244 bp (包含A尾巴)。基于ExPASy、Signal 4.1、TMHMM、SMART等在线软件对Os-vasa基因的蛋白质结构进行预测，得出其氨基酸分子量为76 580.53 Da，理论等电点为5.89。无信号肽，跨膜区域没有明显的信号，因此，推测其为胞内蛋白，不属于膜蛋白。该蛋白具有DEXDc和HELICc 2个功能结构域，而且有9个DEAD-box家族蛋白的典型保守区域，表明所得cDNA属于vasa基因家族。使用qRT-PCR对中华蛸各时期胚胎、初孵幼体及2个发育时期的卵巢及雌雄不同组织的表达模式进行分析。结果显示，Os-vasa基因在性腺中特异性表达，且在卵巢中的表达大于精巢，未成熟和成熟卵巢中均有vasa mRNA表达，且未成熟期表达量较高。因此，推测Os-vasa基因可能在卵巢发育过程和功能维持等方面起到重要作用。在中华蛸早期胚胎发育阶段，均能检测到Os-vasa基因转录本，前10 d微弱表达，从第13天开始，表达量逐渐上升，至第19天达到最高。在初孵幼体阶段，Os-vasa分别在第8天和第20天出现最低和最高表达量。本研究结果可为中华蛸原始生殖细胞起源、迁移和分化提供理论资料，有助于加深对中华蛸卵巢发育和卵子发生过程的理解。

Abstract:

The vasa gene is a member of the DEAD-box family of proteins and plays a key role in the formation of germ cells in eukaryotes. In this study, we cloned the full length (2438 bp) of Octopus sinensis vasa cDNA (Os-vasa) via rapid amplification of cDNA end (RACE) methods. With an open reading frame (ORF) of 2067 bp, encoding 688 amino acids, a 5′UTR of 128 bp, a 3′UTR of 244 bp, and included an A-tail. Based on ExPASy, Signal4.1, TMHMM, and SMART biological analysis, the ORF encoded a putative protein, with a predicted molecular weight of 76 580.53 Da, and the theoretical isoelectric point was 5.89. No signal peptide site was detected, and there was a significant signal in the transmembrane region; therefore, it was presumed to be an intracellular protein, and not a membrane protein. There were two domains, DEXDc and HELICc, and nine conserved motifs of the DEAD-box family, indicating that the cDNA cloned in this study belonged to the family of vasa. Real-time fluorescence quantitative PCR was used to analyze the expression patterns of the Os-vasa gene at different stages of the embryo and larva, in the ovaries at two growth stages, and in specific tissues for males and females. The results showed that the Os-vasa gene was especially expressed in the gonads, and the expression level in the ovary was significantly higher than that in the testis; vasa mRNA was expressed in both immature and mature ovaries, and the transcript level of the immature stage was evidently higher than the mature stage, revealing that the Os-vasa gene might play an important role in the development process and the maintenance of ovarian functions. Os-vasa gene transcripts were detected at whole embryonic developmental stages, were weakly expressed first 10 days, and gradually increased from the 13th day to the highest level on the 19th day. In the larval stages, vasa exhibited the lowest and highest expression on the 8th day post-hatching and the 20th day, respectively. The findings of this study can provide information for the study of primordial germ cell origin and migration and differentiation, and can contribute to the understanding of ovarian development and oogenesis of O. sinensis.