脊尾白虾水通道蛋白基因4和11在碱度胁迫过程中的作用

The roles of aquaporin gene 4 and 11 of Exopalaemon carinicauda under alkalinity stress

中文关键词: 脊尾白虾  水通道蛋白  基因克隆  渗透压

英文关键词: Exopalaemon carinicauda  Aquaporin  Gene cloning  Osmolality

中文摘要:

      水通道蛋白(aquaporin)是一种细胞膜上特异性转运水分子及其他中性代谢分子的膜蛋白家族，对生物的细胞内外渗透压稳定具有重要的调节作用。为了了解水通道蛋白在脊尾白虾(Exopalaemon carinicauda)应对碱度胁迫中的作用，本研究利用RACE技术成功克隆了脊尾白虾水通道蛋白4 (aquaporin 4, EcAQP4)与水通道蛋白11 (aquaporin 11, EcAQP11)基因cDNA全长，EcAQP4基因的开放阅读框为621 bp，编码206个氨基酸，预测分子量为21.673 kDa，理论等电点为8.30，为疏水性蛋白，具有5个跨膜结构域；EcAQP11基因的开放阅读框长度为783 bp，编码260个氨基酸，预测蛋白分子量为28.490 kDa，理论等电点为5.40，为疏水性蛋白，具有4个跨膜结构域。序列比对结果显示，EcAQP4基因与罗氏沼虾(Macrobrachium rosenbergii)同源性最高，为94.63%；EcAQP11与斑节对虾(Penaeus monodon)同源性最高，为81.47%。为验证水通道蛋白的功能，利用RNA干扰技术特异性沉默EcAQP4和EcAQP11基因，结果显示，碳酸盐碱度胁迫后，注射干扰后的脊尾白虾死亡率显著升高，EcAQP4干扰组72 h死亡率达到45%，EcAQP11干扰组72 h死亡率达到55%，与对照组差异显著(P<0.05)。同时发现，EcAQP4干扰组在碳酸盐碱度胁迫24、48与72 h时的血液渗透压变化幅度显著高于对照组(P<0.05)，72 h时渗透压显著升高(P<0.05)；EcAQP11干扰组血液渗透压在3个时间点均显著升高(P<0.05)。以上结果表明，水通道蛋白在脊尾白虾应对碱度胁迫过程中起到了调节渗透压、维持体内外离子平衡的作用。

英文摘要:

      Aquaporins are a family of cell membrane proteins, and their key role is to specifically transport water molecules and other neutral metabolic molecules. Moreover, aquaporins play an important role in regulating the balance between internal and external osmotic pressure in organisms. Here, aquaporin 4 and 11 genes from Exopalaemon carinicauda were successfully cloned using rapid amplification of cDNA ends cloning. The open reading frame of EcAQP4 is 621 bp, encoding 206 amino acids, with a predicted molecular weight of 21.673 kDa and a theoretical isoelectric point of 8.30; it is a hydrophobic protein with five transmembrane structural domains. The open reading frame of EcAQP11 is 783 bp, encoding 260 amino acids, with a predicted molecular weight of 28.490 kDa and a theoretical isoelectric point of 5.40; it is a hydrophobic protein with four transmembrane domains. In sequence alignment, AQP4 in E. carinicauda shared the highest homology with that in Macrobrachium rosenbergii (94.63%), while AQP11 in E. carinicauda shared the highest homology with that in Penaeus monodon (81.47%). Furthermore, RNA interference was used for silencing EcAQP4 and EcAQP11 expression to verify their function. In carbonate alkalinity stress, the mortality of E. carinicauda increased significantly following RNA interference. At 72 h, mortality reached 45% and 55% in the EcAQP4 and EcAQP11 groups, respectively, being significantly higher than that in the control group. The blood osmotic pressure of E. carinicauda in the EcAQP4 group was significantly higher than that in the control group. Similarly, the blood osmotic pressure of E. carinicauda in the EcAQP11 group was significantly increased. In summary, aquaporins play important roles in regulating osmotic pressure and maintaining ion balance in response to alkalinity stress in E. carinicauda.