黄颡鱼Wnt家族4个基因的克隆、组织表达及对铜的响应

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

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c DNA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m RNA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 m RNA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60μg Cu/L组最低,其他2个组差异不显著,Wnt9b m RNA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。

Four full length Wnt c DNA sequences, designated Wnt5 a, Wnt5 b, Wnt7 a and Wnt9 b, were amplified by RTPCR and RACE approaches from yellow catfish Pelteobagrus fulvidraco. The validated c DNAs of Wnt5 a, Wnt5 b,Wnt7 a and Wnt9 b 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 m RNAs 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 Wnt7 a m RNA levels only at 30 μg Cu/L group. Cu exposure had no impact on m RNA levels of Wnt5 a, Wnt5 b and Wnt9 b at day 28. On 56-day Cu exposure, m RNA levels of Wnt5 b were not lowest at 60 μg Cu/L group and Wnt9 b were the highest at 30 μg Cu/L group. Wnt5 a and Wnt7 a 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.