Huang Jinqiang, Chen Songlin, Liu Yang, Shao Changwei, Lin Fan, Wang Na, Hu Qiaomu
College of Marine Life Sciences, Ocean University of China, Qingdao, China; Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China; College of Animal Science and Technology, Gansu Agricultural University, Lanzhou, China.
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China.
Theriogenology. 2014 Jul 15;82(2):213-24. doi: 10.1016/j.theriogenology.2014.03.017. Epub 2014 Mar 31.
Vasa is a highly conserved ATP-dependent RNA helicase expressed mainly in germ cells. The vasa gene plays a crucial role in the development of germ cell lineage and has become an excellent molecular marker in identifying germ cells in teleosts. However, little is known about the structure and function of the vasa gene in flatfish. In this study, the vasa gene (Csvasa) was isolated and characterized in half-smooth tongue sole (Cynoglossus semilaevis), an economically important flatfish in China. In the obtained 6425-bp genomic sequence, 23 exons and 22 introns were identified. The Csvasa gene encodes a 663-amino acid protein, including highly conserved domains of the DEAD-box protein family. The amino acid sequence also shared a high homology with other teleosts. Csvasa expression was mainly restricted to the gonads, with little or no expression in other tissues. Real-time quantitative polymerase chain reaction analysis revealed that Csvasa expression levels decreased during embryonic and early developmental stages and increased with the primordial germ cell proliferation. A typical sexually dimorphic expression pattern of Csvasa was observed during early development and sex differentiation, suggesting that the Csvasa gene might play a differential role in the proliferation and differentiation of male and female primordial germ cells (PGCs). Csvasa mRNA expression levels in neomales were significantly lower than those in normal males and females, indicating that the Csvasa gene might be implicated in germ cell development after sex reversal by temperature treatment. In addition, medaka (Oryzias latipes) PGCs could be transiently labeled by microinjection of synthesized mRNA containing the green fluorescence protein gene and 3'-untranslated region of Csvasa, which confirmed that the Csvasa gene has the potential to be used as a visual molecular marker of germ cells and laid a foundation for manipulation of PGCs in tongue sole reproduction.
瓦萨蛋白是一种高度保守的依赖ATP的RNA解旋酶，主要在生殖细胞中表达。瓦萨基因在生殖细胞谱系的发育中起着关键作用，已成为硬骨鱼中鉴定生殖细胞的优良分子标记。然而，关于比目鱼中瓦萨基因的结构和功能知之甚少。在本研究中，在中国一种具有重要经济价值的比目鱼——半滑舌鳎（Cynoglossus semilaevis）中分离并鉴定了瓦萨基因（Csvasa）。在所获得的6425 bp基因组序列中，鉴定出23个外显子和22个内含子。Csvasa基因编码一个663个氨基酸的蛋白质，包括DEAD盒蛋白家族的高度保守结构域。该氨基酸序列与其他硬骨鱼也具有高度同源性。Csvasa的表达主要局限于性腺，在其他组织中很少或没有表达。实时定量聚合酶链反应分析表明，Csvasa的表达水平在胚胎和早期发育阶段下降，并随着原始生殖细胞的增殖而增加。在早期发育和性别分化过程中观察到Csvasa典型的性二态性表达模式，表明Csvasa基因可能在雄性和雌性原始生殖细胞（PGCs）的增殖和分化中发挥不同作用。新雄性个体中Csvasa mRNA表达水平显著低于正常雄性和雌性个体，表明Csvasa基因可能与温度处理导致的性别逆转后的生殖细胞发育有关。此外，通过显微注射含有绿色荧光蛋白基因和Csvasa 3'非翻译区的合成mRNA，可以短暂标记青鳉（Oryzias latipes）的PGCs，这证实了Csvasa基因有潜力用作生殖细胞的可视化分子标记，并为舌鳎繁殖中PGCs的操作奠定了基础。
