花鲈aqp3a基因的克隆、表达分析及渗透调节功能研究

Isolation and Expression Analysis of Spotted Sea Bass(Lateolabrax maculatus)aqp3a and Deduction of Its Osmotic Regulation Function

水通道蛋白3(AQP3)是水通道蛋白家族成员之一,在鱼类的渗透调节过程中发挥着重要作用。本研究从花鲈(Lateolabrax maculatus)中克隆出aqp3a基因,其mRNA序列长度为2058 bp,编码302个氨基酸,含有6个α跨膜螺旋和2个水通道蛋白家族的特征性天冬酰胺-脯氨酸-丙氨酸(NPA)序列。多重序列比对分析表明aqp3a基因具有高度保守性。采用枝位点模型对aqp3进行选择压力分析,结果显示aqp3在进化过程中存在4个显著的正选择位点。qRT-PCR检测结果表明,花鲈aqp3a基因在其各组织中广泛表达,且在鳃中表达显著高于其它组织。此外,还检测分析了花鲈aqp3a基因在淡水和海水驯化过程中于各时间点(0 h、12 h、1 d、3 d、7 d)在鳃组织中的表达情况。结果显示:在淡水适应过程中,花鲈aqp3a基因的表达量呈先升后降趋势,1 d时表达量显著上调并达到最大值,7 d时的表达量降低至与淡水转化前无显著差异的水平。在海水适应过程中,aqp3a基因在鳃中的表达量逐渐下降然后维持稳定。将花鲈aqp3a基因的cRNA显微注射进非洲爪蟾(Xenopus laevis)的卵母细胞中进行异源表达,卵母细胞在5 min内膨胀至初始体积的1.3~1.4倍,透水系数(Pf)约为注射蒸馏水对照组的5倍,表明花鲈aqp3a基因在水的转运和细胞体积调节中发挥着重要作用。本研究为深入了解花鲈aqp3a基因的渗透调节功能提供了理论基础,同时为其它鱼类渗透调节相关功能基因的研究提供参考。

Aquaporin 3(AQP3)is a member of aquaporin family and plays important roles in the osmotic regulation of fish.In this study,aqp3a gene was isolated from Lateolabrax maculatus.The full length of its cDNA sequence was 2058 bp,encoding 302 amino acids.The deduced protein contained 6αtransmembrane helices and 2 characteristic Aspartame-Proline-Alanine(NPA)sequences of aquaporin families.Multiple sequence alignment analysis showed that aqp3a was highly conserved.The branch site model was used to analyze the selection pressure of aqp3.The results showed that there were 4 significant positive selection sites in the evolution process of aqp3.qRT-PCR results showed that aqp3a widely expressed in tested tissues,and the expression level in gills was significantly higher than that in other tissues.In addition,the expression of aqp3a in the gill of spotted sea bass during freshwater and seawater acclimation(0 h,12 h,1 d,3 d,7 d)was analyzed.The results showed that in the process of freshwater adaptation,the expression level of aqp3a increased firstly and then decreased.At 1d,the expression level was significantly up-regulated and reached its maximum value,while on day 7,the expression level was reduced to the same value as it was on day 0.During seawater adaptation,the expression of aqp3a in gills decreased gradually and then remained stable.In addition,the aqp3a cRNA of spotted sea bass was injected into the oocyte of Xenopus laevis by microinjection.The oocytes swelled to 1.3~1.4 times of the initial volume within 5 minutes,with a permeable coefficient(Pf)about 5 times of the control group,indicating that spotted sea bass aqp3a may play important roles in water transport and regulation of cell volume.This study laid a theoretical basis for further understanding the osmotic regulation function of aqp3a in spotted sea bass,and provided a reference for studies of osmoregulation related functional genes in other fish species.