Li Hailong, Zhang Zhifeng, Bi Ying, Yang Dandan, Zhang Litao, Liu Jianguo
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao, China; Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China.
Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao, China.
PLoS One. 2014 Dec 30;9(12):e115917. doi: 10.1371/journal.pone.0115917. eCollection 2014.
β-catenin is a key signaling molecule in the canonical Wnt pathway, which is involved in animal development. However, little information has been reported for β-catenin in bivalves. In the present study, we cloned a homolog of β-catenin from the scallop Chlamys farreri and determined its expression characteristics. The full-length cDNA of β-catenin was 3,353 bp, including a 2,511 bp open reading frame that encoded a predicted 836 amino acid protein. Level of the β-catenin mRNA increased significantly (P<0.05) with C. farreri gonadal development and presented a sexually dimorphic expression pattern in the gonads, which was significantly high in ovaries detected by quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical analysis revealed that the β-catenin was mainly located in germ cells of the gonads, with obvious positive immune signals in the oogonia and oocytes of ovaries as well as in the spermatogonia and spermatocytes of testes, implying β-catenin might be involved in the gametogenesis of C. farreri. Furthermore, when 0.1 µg/mL and 0.2 µg/mL DKK-1 (an inhibitor of the canonical Wnt pathway) were added in vitro to culture medium containing testis cells of C. farreri, the expression of β-catenin decreased significantly detected by qRT-PCR (P<0.05), suggesting the canonical Wnt signal pathway exists in the scallop testis. Similarly, when 50 µM and 100 µM quercetin (an inhibitor of β-catenin) were added in vitro to the culture system, Dax1 expression was significantly down-regulated compared with controls (P<0.05), implying the β-catenin is an upstream gene of Dax1 and is involved in the regulation of C. farreri spermatogenesis.
β-连环蛋白是经典Wnt信号通路中的关键信号分子，参与动物发育过程。然而，关于双壳贝类中β-连环蛋白的报道较少。在本研究中，我们从栉孔扇贝中克隆了β-连环蛋白的同源物，并确定了其表达特征。β-连环蛋白的全长cDNA为3353 bp，其中包括一个2511 bp的开放阅读框，编码一个预测的836个氨基酸的蛋白质。随着栉孔扇贝性腺发育，β-连环蛋白mRNA水平显著升高（P<0.05），并且在性腺中呈现出性别二态性表达模式，通过定量实时聚合酶链反应（qRT-PCR）检测发现其在卵巢中的表达显著高于睾丸。免疫组织化学分析表明，β-连环蛋白主要位于性腺的生殖细胞中，在卵巢的卵原细胞和卵母细胞以及睾丸的精原细胞和精母细胞中具有明显的阳性免疫信号，这意味着β-连环蛋白可能参与了栉孔扇贝的配子发生。此外，当在含有栉孔扇贝睾丸细胞的培养基中体外添加0.1 μg/mL和0.2 μg/mL的DKK-1（经典Wnt信号通路的抑制剂）时，通过qRT-PCR检测发现β-连环蛋白的表达显著降低（P<0.05），表明栉孔扇贝睾丸中存在经典Wnt信号通路。同样，当在体外培养体系中添加50 μM和100 μM的槲皮素（β-连环蛋白抑制剂）时，与对照组相比，Dax1的表达显著下调（P<0.05），这意味着β-连环蛋白是Dax1的上游基因，参与了栉孔扇贝精子发生的调控。
