花鳗鲡AQP3基因的分子特征及其应对盐度变化的表达规律

Cloning and effects of different salinities on aquaporin 3(AQP3) in juvenile marbled eel,Anguilla marmorata

为了探究花鳗鲡(Anguila marmorata)AQP3基因在不同盐度下的表达规律,利用RACE技术克隆了花鳗鲡AQP3基因c DNA全长序列,并进行了生物信息学分析;通过实时荧光定量PCR(RT-q PCR),检测了花鳗鲡AQP3在9个组织中的分布及盐度胁迫后鳃、肾、肠组织在0、6、12、24 h和3、5、10 d的表达情况。结果显示:花鳗鲡AQP3基因的c DNA全长为1299 bp,开放阅读框(ORF)为891 bp,5’非编码区(UTR)和3’非编码区(UTR)分别为60 bp和348 bp,共编码296个氨基酸,具有6段跨膜结构域及2个NPA(天冬氨酸-脯氨酸-丙氨酸)保守结构域。氨基酸多重序列比对结果显示,花鳗鲡AQP3基因与其同属的欧洲鳗鲡(Anguilla anguilla)、日本鳗鲡(Anguilla japonica)同源性分别高达97.0%和96.0%。RT-q PCR检测发现,AQP3基因在鳃组织中表达量最高,而在脾脏中表达量最低;在咸淡水(盐度10)和海水(盐度25)组中,鳃组织的AQP3基因表达水平呈下降趋势,且在各时段均显著下调;肾组织的AQP3表达水平在咸淡水(除24 h外)和海水(除6 h外)组中也显著下调;肠组织的AQP3表达水平在咸淡水组中显著上调,而在海水组中则显著下调。研究表明,花鳗鲡AQP3的mRNA表达量在应对不同盐度环境时呈现不同的表达变化模式,这将为深入揭示花鳗鲡适应盐度变化的分子调控机理奠定理论基础。

In order to successfully adapt to threats of salinities,fish possesses AQPs（aquaporin） which are essential for water transport in the process of osmoregulation and maintaining homeostasis. AQP3 is unique in that it can not only transport water,but also participate in glycerin,urea and other small nitrogen-containing molecule transport activities. The method of RACE was applied to clone the DNA sequence of AQP3 from Anguila marmorata and then bioinformatics analysis was carried on for the purpose of investigating the role of AQP3 in A. marmorata osmotic adjustment process. The technique of real-time qRCR was adopted to detect mRNA expression in the blank tissues（including brain,gill,intestine,kidney,liver,muscle,heart,spleen and swim bladder） of fish acclimated to fresh water（0 ppt）. The same batch of A. marmorata was acclimated to two other salinity gradients： brackish water（10 ppt） and sea water（25 ppt）,to observe expression changing patterns at different time（0 h,6 h,12 h,24 h,3 d,5 d and 10 d） in three osmoregulation associated tissues（including gill,kidney and intestine）. The results showed that the full length of A. marmorata AQP3 c DNA was 1 299 bp,containing an 891 bp open reading frame,with a 60 bp5＇untranslated region（UTR） and 348 bp 3＇UTR. It encoded 296 amino acids,owning a six transmembrane structure and two NPA（aspartic acid-proline-alanine） conserved domains. The multiple amino acid sequence comparison showed that AQP3 of A. marmorata shared the highest identity with European eel（Anguila anguila,97 %） and Japanese eel（Anguila japonica,96 %）. The higher expression was detected in gill,with the highest expression,intestine and muscle,yet an extremely low expression was observed in spleen,with the lowest expression, kidney, liver and other tissues. AQP3 mRNA expression level in the gill decreased significantly at all time of the experiment when exposed to brackish water（10 ppt） and sea water（25 ppt）. In the kidney,the mRNA expression significantly decreased except for 24 h in brackish water and6 h in sea water. In the intestine,the mRNA expression was upregulated in the brackish water group,while the expression in the sea water group was downregulated. The experimental results suggested that AQP3 of A.marmorata may involve in the osmoregulation activity and present different expression changing patterns under different salinity gradients. These findings might contribute a lot to reveal the molecular mechanism of A.marmorata under different salinity gradients.