黄颡鱼Wnt/β-catenin信号通路5个重要基因的识别及其在卵巢中的表达对铜暴露的响应

Identification of five genes from Wnt/β-catenin pathway inyellow catfish (Pelteobagrus fulvidraco) and their mRNA expression inthe ovary to waterborne copper exposure

摘要:

采用RT-PCR和RACE技术获取了黄颡鱼Wnt/β-catenin信号通路5个重要基因wnt2、wnt2bb、wnt3a、wnt8a和ctnnb1的全长cDNA序列，分别为1 743、2 133、1 379、1 508和2 636 bp，其中ORF长度分别为1 052、1 115、872、1 160和2 372 bp，分别编码351、372、291、387和791个氨基酸。氨基酸序列比对和系统发育树分析显示，上述基因十分保守，黄颡鱼与墨西哥丽脂鲤亲缘关系最近。组织表达分析显示，上述基因的mRNA在脑、脾脏、肾脏、鳃、心脏、肌肉、肠系膜脂肪、肝脏及卵巢等组织中都有表达，但表达水平不尽相同。这些基因在黄颡鱼卵巢中的表达水平对铜暴露的响应结果显示，在暴露28 d时，wnt3a mRNA水平随着铜浓度的增加而降低，而wnt8a趋势则相反，wnt2、wnt2bb和ctnnb1基因表达各个处理组间无显著性差异；在56 d时，wnt2、wnt2bb、wnt3a、wnt8a和ctnnb1基因表达各个处理组间均无显著差异。研究表明，Wnt/β-catenin信号通路基因的功能发生了分化，部分成员可能介导了铜影响黄颡鱼卵巢发育的调控。

关键词:黄颡鱼;Wnt/β-catenin;基因克隆;组织;铜暴露

Abstract:

Five complete cDNA sequences related to Wnt/β-catenin signal pathway, designated wnt2, wnt2bb, wnt3a, wnt8a and ctnnb1, were amplified by RT-PCR and RACE approaches from yellow catfish (Pelteobagrus fulvidraco). The validated cDNAs encoding wnt2, wnt2bb, wnt3a, wnt8a and ctnnb1 were 1 743, 2 133, 1 379, 1 508 and 2 636 bp in length, encoding peptides of 351, 372, 291, 387 and 791 amino acid residues, respectively. The amino acid alignment and phylogenetic analysis revealed that these sequences were relatively conserved among vertebrates, and Wnt/β-catenin genes of P. fulvidraco were more closely related to those of Astyanax mexicanus. All Wnt/β-catenin mRNAs were expressed in nine tested tissues (brain, spleen, kidney, gill, heart, muscle, mesenteric fat, liver and ovary), but at variable levels. Effect of waterborne Cu exposure on ovarian mRNA levels of the five genes of P. fulvidraco were determined. On day 28, wnt3a mRNA levels in the ovary significantly decreased with increasing waterborne Cu concentration, but wnt8a showed the opposite trend. On day 56, mRNA levels of these five genes from the ovary of P. fulvidraco showed no significant differences among the three groups. The present study may be helpful to understand the physiological functions of Wnt/β-catenin pathway in fish.

Key words:Pelteobagrus fulvidraco;Wnt/β-catenin;gene cloning;tissue;copper exposure