Molecular cloning and characterization of three novel Hemocyanins from Chinese mitten crab, Eriocheir sinensis
Molecular cloning and characterization of three novel Hemocyanins from Chinese mitten crab, Eriocheir sinensis
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中华绒螯蟹三种新型血蓝蛋白的分子克隆及表征
DOI：
10.1016/j.aquaculture.2014.07.033
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发表时间：
2014-10-20
期刊：
影响因子：
4.5
通讯作者：
Wang, Wen
中科院分区：
文献类型：
--
作者：
Huang, Ying;Huang, Xin;Wang, Wen
血蓝蛋白是一种铜结合蛋白，在甲壳动物的生理过程中起着至关重要的作用。然而，人们对中华绒螯蟹的血蓝蛋白知之甚少。本研究利用表达序列标签(EST)分析和快速扩增基因末端扩增(RACE)技术，从中华绒毛虫中克隆了三种形式的血蓝蛋白，分别命名为EsHc1、EsHc2和EsHc3。EsHc1、EsHc2和EsHc3基因的开放阅读框(ORF)分别为2182、2580和2220bp，编码678、662和691个氨基酸，均含有3个串联的血蓝蛋白结构域：1)血蓝蛋白N，2)血蓝蛋白M或酪氨酸酶，3)血蓝蛋白C结构域。BLASTP和系统发育树分析表明，EsHc1与PpCc1和MMCC聚为一类，EsHc2和EsHc3与PIHc和MmHc1聚在一起。EsHc1主要在肝胰腺和血细胞中表达，在神经、眼柄、肌肉、肠、鳃和心脏中表达水平较低，而EsHc2和EsHc3主要在肝胰腺表达，在血细胞中也有表达。实时荧光定量RT-PCR分析显示，在脂多糖(LPS)、肽聚糖(PGN)、金黄色葡萄球菌、副溶血性弧菌和嗜水气单胞菌感染后的不同时间点，肝胰腺中EsHcs基因的转录均有显著表达。综上所述，有证据表明，血蓝蛋白基因的三个亚型参与了针对中华绒螯蟹感染细菌的先天免疫反应。(C)2014爱思唯尔B.V.保留所有权利。
Hemocyanin is a copper-binding protein and plays a crucial role in the physiological processes in crustaceans. However, little is known about the hemocyanin from the Chinese mitten crab, Eriocheir sinensis. In this study, three forms of hemocyanins designated as EsHc1, EsHc2 and EsHc3 were cloned from E. sinensis by using expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The open reading frames (ORFs) of EsHc1, EsHc2 and EsHc3 genes were 2182, 2580 and 2220 bp encoding proteins with 678, 662 and 691 amino acids, respectively, and all contain three tandem hemocyanin domains: 1) hemocyanin N, 2) hemocyanin M or tyrosinase, and 3) hemocyanin C domain. BLASTP and phylogenetic tree analysis showed that EsHc1 was clustered together with cryptocyanin I from Portunus pelagicus (PpCc1) and cyptocyanin from Metacarcinus magister (MmCc), EsHc2 and EsHc3 with hemocyanin from Pacifastacus leniusculus (PIHc) and hemocyanin subunit 1 from M. magister (MmHc1) were gathered into one clade. EsHc1 was mainly expressed in hepatopancreas and hemocytes with a lower level of expression in nerves, eyestalk, muscles, intestine, gills and heart; whereas EsHc2 and EsHc3 were mainly expressed in hepatopancreas and were also detected in hemocytes. Quantitative real-time RT-PCR analysis showed that EsHcs mRNA transcription in hepatopancreas was significantly expressed at various time points after infection with Lipopolysaccharide (LPS), peptidoglycan (PGN), Staphylococcus aureus, Vibrio parahaemolyticus and Aeromonas hydrophila. In summary, there is evidence that the three isoforms of hemocyanin genes participate in the innate immune response against bacteria infecting the Chinese mitten crab. (C) 2014 Elsevier B.V. All rights reserved.