| 摘要: |
| 采用RACE技术获得了总长为1853bp的脊尾白虾(Exopalaemon carinicauda)组织蛋白酶D基因全长cDNA序列。该基因5′和3′非编码区分别为120bp和572bp, 开放阅读框为1161bp, 推测编码386个氨基酸, 预测分子量为42.36kDa, 理论等电点为7.54, 命名为EcCatD基因。同源性和系统进化分析表明, EcCatD基因与斑节对虾、美洲螯龙虾的相似性分别为86%和80%, 与斑节对虾和美洲螯龙虾紧密聚为一支。荧光定量RT-PCR结果表明, EcCatD基因在血细胞中的相对表达量最高, 其次为肝胰腺。该基因在鳗弧菌和WSSV感染后的脊尾白虾血细胞和肝胰腺中的表达量显著增加, 并具有不同的时空表达趋势。结果表明EcCatD基因在脊尾白虾免疫反应中具有重要作用。 |
| 关键词: 脊尾白虾, 组织蛋白酶D, 基因克隆, 表达 |
| DOI：10.11693/hyhz201303009009 |
| 分类号: |
| 基金项目:国家高技术研究发展计划课题, 2012AA10A409 号; 国家虾产业技术体系, CARS-47 号; 公益性行业(农业)科研专项课题, 201103034号; 中国水产科学研究院基本科研业务费资助, 2013A0701 号 |
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| CLONING AND EXPRESSION OF CATHEPSIN D GENE IN EXOPALAEMON CARINICAUDA |
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DUAN Ya-Fei1,2, LIU Ping1, LI Ji-Tao1, LI Jian1, CHEN Ping1, GAO Bao-Quan1, XU Wen-Fei1,3, WANG You-Kun1,2
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1.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences;2.Ocean University of Shanghai;3.Ocean University of Dalian
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| Abstract: |
| The cathepsin D gene (CatD) of Exopalaemon carinicauda was first cloned using RACE method, which was named EcCatD. The full length cDNA sequence of CatD is 1853 bp, which contains a 120 bp 5′-UTR (untranslated region), 572 bp 3′-UTR, and 1161 bp open reading frame (ORF) that encodes 386 amnio-acid polypeptides, which has the isoelectric point (pI) of 7.54 and molecular mass of 42.36 kDa. Blast analysis revealed that the dentifies of EcCatD with other crustacean species were over 80%, such as Penaeus monodon 86% and Homarus americanus 80%, indicating that this protein was one of cathepsin D CatD family members. The phylogenetic analysis using neighbor-joining based on amino acid sequences showed that E. carinicauda EcCatD was in the same category as CatD of P. monodon and H. americanus. The expression level of EcCatD gene in different tissues were analyzed by quantitative Real-time PCR, the results showed that the highest expression level of EcCatD gene was in hemocytes, and the hepatopancreas was the second. Real-time PCR analysis showed that the expression level of EcCatD was up-regulated distinctly in the hemocytes and hepatopanereas of E. carinicauda after challenged by Vibrio anguillarum and white spot syndrome virus (WSSV). EcCatD showed different expression profiles in the hemocytes and hepatopancreas during V. anguillarum or WSSV virus infection. The results implied that EcCatD might play an important role in the prawn immune system. |
| Key words: Exopalaemon carinicauda Cathepsin D Gene cloning Expression |
