Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, PR China.
Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, PR China; Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, PR China.
Fish Shellfish Immunol. 2016 Aug;55:123-30. doi: 10.1016/j.fsi.2016.05.028. Epub 2016 May 24.
ADP ribosylation factors (Arf), as highly conserved small guanosine triphosphate (GTP)-binding proteins, participates in intracellular trafficking and organelle structure. In this study, a full-length cDNA of Arf1 (designated EcArf1) was cloned from Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EcArf1 was 1428 bp, which contains an open reading frame (ORF) of 549 bp, encoding a 182 amino-acid polypeptide with the predicted molecular weight of 20.69 kDa and estimated isoelectric point was 7.24. Sequence analysis revealed that the conserved Arf protein family signatures were identified in EcArf1. The deduced amino acid sequence of EcArf1 shared high identity (95%-98%) with that of other species and clustered together with Arf1 of other shrimp in the NJ phylogenetic tree, indicating that EcArf1 should be a member of the Arf1 family. Quantitative real-time RT-qPCR analysis indicated that EcArf1 was expressed in hemocytes, hepatopancreas, gills, muscle, ovary, intestine, stomach and heart, and the most abundant level was in hemocytes and gills, which were also the two main target tissues of pathogen infection and environmental stress. After Vibrio parahaemolyticus challenge, EcArf1 transcripts level significantly increased in hemocytes and hepatopancreas at 3 h and 6 h, respectively. The expression of EcArf1 in hemocytes and hepatopancreas significantly up-regulated at 12 h and 6 h respectively, and down-regulated at 72 h and 48 h, respectively. EcArf1 expression in hepatopancreas and gills both significantly increased at 6 h and decreased at 24 h under ammonia-N stress. The results suggested that EcArf1 might be involved in immune responses to pathogens (V. parahaemolyticus and WSSV) challenge and ammonia-N stress in E. carinicauda.
ADP 核糖基化因子 (Arf) 作为高度保守的小 G 蛋白三磷酸鸟苷 (GTP) 结合蛋白，参与细胞内运输和细胞器结构。在这项研究中，通过快速扩增 cDNA 末端 (RACE) 方法，从中国对虾中克隆了全长 cDNA 的 Arf1（命名为 EcArf1）。EcArf1 的全长 cDNA 为 1428 bp，包含一个 549 bp 的开放阅读框 (ORF)，编码一个 182 个氨基酸的多肽，预测分子量为 20.69 kDa，估计等电点为 7.24。序列分析表明，EcArf1 中存在保守的 Arf 蛋白家族特征。EcArf1 的推导氨基酸序列与其他物种高度一致（95%-98%），并与其他虾类的 Arf1 在 NJ 系统发育树中聚类在一起，表明 EcArf1 应该是 Arf1 家族的成员。定量实时 RT-qPCR 分析表明，EcArf1 在血细胞、肝胰腺、鳃、肌肉、卵巢、肠、胃和心脏中表达，最丰富的水平在血细胞和鳃中，这也是病原体感染和环境胁迫的两个主要靶组织。在副溶血弧菌攻毒后，EcArf1 在血细胞和肝胰腺中的转录水平分别在 3 h 和 6 h 显著增加。EcArf1 在血细胞和肝胰腺中的表达分别在 12 h 和 6 h 显著上调，在 72 h 和 48 h 显著下调。在氨氮胁迫下，EcArf1 在肝胰腺和鳃中的表达均在 6 h 时显著增加，在 24 h 时显著下降。结果表明，EcArf1 可能参与了中国对虾对病原体（副溶血弧菌和 WSSV）攻毒和氨氮胁迫的免疫反应。
