脊尾白虾丝氨酸蛋白酶抑制剂基因克隆及表达分析

采用RACE方法获得了脊尾白虾(Exopalaemon carinicauda)丝氨酸蛋白酶抑制剂serpin基因的cDNA序列。该基因全长1 516 bp, 开放阅读框1 245 bp, 编码414个氨基酸, 其预测分子量为45.06 kD, 理论等电点为5.694, 并含有两个糖基结合位点。同源性分析发现其与斑节对虾(Penaeus monodon)同源性最高, 达到49%。组织表达分析表明, serpin基因在脊尾白虾血细胞中表达量最高, 其次是肝胰腺和鳃组织, 在肌肉中表达量最低。不同盐度胁迫后脊尾白虾的血细胞serpin基因表达量在盐度胁迫8 h时显著高于对照组(P<0.05), 肝胰腺组织中serpin基因表达量在盐度胁迫后2 h和24 h出现明显峰值, 且显著高于对照组(P<0.05)。上述结果表明, 脊尾白虾serpin基因参与了急性盐度胁迫下机体的应激反应。本研究通过克隆脊尾白虾酚氧化酶原激活系统中的丝氨酸蛋白酶抑制剂基因cDNA全长, 分析盐度胁迫后其在血细胞和肝胰腺组织中的表达变化规律, 以期为脊尾白虾的健康养殖提供理论基础和参考依据。

Cloning and expression analysis of serine protease inhibitors gene of Exopalaemon carinicauda

Exopalaemon carinicauda belongs to Palaemonidae, Palaemon, and Exopalaemon, which is an important demersal species in the Yellow Sea and Bohai Sea. Its production ranks only second to that of Fenneropenaeus chinensiss and Acetes chinensis in China. This species is mainly charactered by its tender meat, high reproduction, fast growth and adaption to a wide range of environment. Recently, the aquaculture area has been expanded gradually, but under the condition of centralization-breeding factor, the shrimp is easy in stressing conditions, which may cause diseases even death. In this study, we isolated the cDNA sequence of serine protease inhibitors (serpin) gene from Exopalaemon carinicauda using RT-PCR and RACE methods. The full-length cDNA of the serine protease consisted of 1 516 bp with a 1 245 bp open reading frame (ORF), encoding 414 amino acids. The predicted molecular mass of serine protease protein was 45.06 kD, and the theoretical isoelectric point was 5.694. The serine protease protein sequence contained two glycosyl binding sites, which had 49% homology with that of Penaeus monodon. Tissue expression analysis suggested that serpin mRNA expression was highest in the hemocytes and lowest in the muscle. Real time-PCR analysis revealed that the expression of serpin gene was significantly higher in hemolymph 8 h after salinity exposure compared to the control group. Furthermore, the serpin gene expression in the hepatopancreas exhibited two obvious peaks at 2 h and 24 h after salinity stress compared with the control group. Our results suggest that serpin gene plays an important role in the salinity stress response of E. carinicauda.