黄颡鱼Wnt家族4个基因的克隆、组织表达及对铜的响应

为探究Wnt家族成员在黄颡鱼卵巢发育中的作用, 研究首先采用RT-PCR和RACE技术获取了黄颡鱼Wnt5a、Wnt5b、Wnt7a和Wnt9b基因的全长cDNA序列, 即1984、2905、2158和1622 bp, 其中ORF长度分别为1124、1124、1049和1073 bp, 编码375、375、350和358个氨基酸。氨基酸序列比对和系统树分析显示, 这些基因十分保守, 黄颡鱼WNT与墨西哥丽脂鲤和斑马鱼比较接近。组织表达分析表明, 这些基因的mRNA在脑、脾脏、肾脏、鳃、心脏、肌肉、脂肪、肝脏及卵巢等组织中都有表达, 但表达水平不尽相同。Wnt家族基因对铜的响应研究表明: 在暴露28d时, Wnt7a mRNA水平随着铜浓度先上升后下降, 但是Wnt5a、Wnt5b和Wnt9b基因表达各个处理组无显著性差异; 在56d, Wnt5b在60 μg Cu/L组最低, 其他2个组差异不显著, Wnt9b mRNA水平随着铜浓度先上升后下降, 但是Wnt5a和Wnt7a基因表达各个处理组无显著性差异, 表明Wnt家族这些基因的功能发生了分化, 部分成员介导了铜影响黄颡鱼卵巢发育的调控。研究首次揭示了黄颡鱼Wnt家族部分成员的基因结构和功能, 为深入探讨他们在卵巢发育中的作用奠定了基础。

CLONING AND TISSUE EXPRESSION OF FOUR WNT GENES IN YELLOW CATFISH PELTEOBAGRUS FULVIDRACO AND THEIR RESPONSE TO COPPER EXPOSURE

Four full length Wnt cDNA sequences, designated Wnt5a, Wnt5b, Wnt7a and Wnt9b, were amplified by RT-PCR and RACE approaches from yellow catfish Pelteobagrus fulvidraco. The validated cDNAs of Wnt5a, Wnt5b, Wnt7a and Wnt9b were 1984, 2905, 2158 and 1622 bp in length, encoding peptides of 375, 375, 350 and 358 amino acid residues, respectively. The amino acid alignment and phylogenetic analysis revealed that Wnt sequences were relatively conserved among different vertebrates. All Wnt mRNAs were expressed in tested tissues (brain, spleen, kidney, gill, heart, muscle, mesenteric fat, liver and ovary) but with variable levels. Cu exposure for 28 days highly induced Wnt7a mRNA levels only at 30 μg Cu/L group. Cu exposure had no impact on mRNA levels of Wnt5a, Wnt5b and Wnt9b at day 28. On 56-day Cu exposure, mRNA levels of Wnt5b were not lowest at 60 μg Cu/L group and Wnt9b were the highest at 30 μg Cu/L group. Wnt5a and Wnt7a showed no significant differences among three groups. The present study indicated that functional differentiation exists in members of Wnt family, and some members may mediate the effect of Cu exposure on ovary development of yellow catfish.