脊尾白虾血蓝蛋白基因cDNA的克隆与表达分析

cDNA cloning and expression analysis of hemocyanin gene in Exopalaemon carinicauda

中文关键词: 脊尾白虾  血蓝蛋白  基因克隆  表达分析

英文关键词: Exopalaemon carinicauda  Hemocyanin  Cloning  Expression

中文摘要:

      应用RACE技术克隆了脊尾白虾血蓝蛋白基因全长cDNA序列，并对该序列进行了分析。结果显示，该基因全长2 158 bp，开放式阅读框长1 992 bp，5’非编码区长26 bp，3’非编码区长140 bp，将该基因命名为EcHc。EcHc编码663个氨基酸，前15个氨基酸组成信号肽，推测成熟肽的分子量为75.05kDa。Blast比对结果显示，由脊尾白虾血蓝蛋白cDNA序列推导的氨基酸序列与日本沼虾、信号小龙虾血蓝蛋白氨基酸序列的同源性分别为86%、68%，由此推断该cDNA序列可能属于血蓝蛋白家族。利用Real-time PCR方法，分别研究了鳗弧菌和白斑综合征病毒 (WSSV)感染脊尾白虾后，肝胰腺组织中EcHc基因在不同时间点的表达变化。实验结果表明，细菌或病毒感染后，EcHc基因在肝胰腺组织中的表达量显著增加，且均在感染后6 h达到最高值，揭示脊尾白虾血蓝蛋白基因EcHc在脊尾白虾的免疫防御中具有重要作用。

英文摘要:

      In this study, a hemocyanin cDNA named EcHc was cloned from the hepatopancreas of the ridgetail white prawn Exopalaemon carinicauda by reverse transcription PCR (RT PCR) and rapid amplification of cDNA end (RACE) methods. The full length cDNA of EcHc was 2 158 bp and had an open reading frame(ORF) of 1 992bp encoding 663 amino acids which contained a signal peptide sequence(1~15 amino acid residues). The predicted molecular weight of the mature peptide was 75.05kDa, and the theoretical pI was 569. The homology analysis of EcHc with other hemocyanin showed higher similarity with Macrobrachium nipponense (86%). Quantitative real time RT PCR analysis revealed that microbial challenge resulted in mRNA up regulation, up to 6h post injection of Vibrio anguillarum or white spot syndrome virus (WSSV). The results indicated that the EcHc plays important roles in the immune defense system of E. carinicauda.