Wang Zhongkai, Gao Jinning, Song Huayu, Wu Xiaomeng, Sun Yan, Qi Jie, Yu Haiyang, Wang Zhigang, Zhang Quanqi
Key Laboratory of Marine Genetics and Breeding (MGB), Ministry of Education, College of Marine Life Sciences, Ocean University of China, Qingdao, China.
PLoS One. 2014 Mar 26;9(3):e93380. doi: 10.1371/journal.pone.0093380. eCollection 2014.
The vasa gene encodes an ATP-dependent RNA helicase of the DEAD box protein family that functions in a broad range of molecular events involving duplex RNA. In most species, the germline specific expression of vasa becomes a molecular marker widely used in the visualization and labeling of primordial germ cells (PGCs) and a tool in surrogate broodstock production through PGC transplantation. The vasa gene from tongue sole (Cynoglossus semilaevis) was characterized to promote the development of genetic breeding techniques in this species. Three C. semilaevis vasa transcripts were isolated, namely vas-l, vas-m, and vas-s. Quantitative real-time PCR results showed that C. semilaevis vasa transcripts were prevalently expressed in gonads, with very weak expression of vas-s in other tissues. Embryonic development expression profiles revealed the onset of zygotic transcription of vasa mRNAs and the maternal deposit of the three transcripts. The genetic ZW female juvenile fish was discriminated from genetic ZZ males by a pair of female specific primers. Only the expression of vas-s can be observed in both sexes during early gonadal differentiation. Before PGCs started mitosis, there was sexually dimorphic expression of vas-s with the ovary showing higher levels and downward trend. The results demonstrated the benefits of vasa as a germline specific marker for PGCs during embryonic development and gonadal differentiation. This study lays the groundwork for further application of C. semilaevis PGCs in fish breeding.
瓦萨基因编码一种DEAD盒蛋白家族的ATP依赖性RNA解旋酶，其在涉及双链RNA的广泛分子事件中发挥作用。在大多数物种中，瓦萨基因的生殖系特异性表达成为一种广泛用于原始生殖细胞（PGC）可视化和标记的分子标记，以及通过PGC移植生产替代亲鱼的工具。对半滑舌鳎（Cynoglossus semilaevis）的瓦萨基因进行了表征，以促进该物种遗传育种技术的发展。分离出了三种半滑舌鳎瓦萨转录本，即vas-l、vas-m和vas-s。实时定量PCR结果表明，半滑舌鳎瓦萨转录本在性腺中普遍表达，vas-s在其他组织中的表达非常弱。胚胎发育表达谱揭示了瓦萨mRNA合子转录的起始以及这三种转录本的母源沉积。通过一对雌性特异性引物将遗传型ZW雌性幼鱼与遗传型ZZ雄性区分开来。在性腺早期分化过程中，两性中均能观察到vas-s的表达。在PGC开始有丝分裂之前，vas-s存在性二态性表达，卵巢中的表达水平较高且呈下降趋势。结果证明了瓦萨作为胚胎发育和性腺分化过程中PGC的生殖系特异性标记的优势。本研究为半滑舌鳎PGC在鱼类育种中的进一步应用奠定了基础。
