关键词: 血蓝蛋白, 基因克隆, cDNA末端快速扩增, 实时荧光定量PCR, 时空表达, 日本沼虾

Abstract: Objective The aim of this study is to clone the hemocyanin gene from EM>Macrobrachium nipponense/EM>,and to investigate the bioinformatics and spatial and temporal expression analysis of the gene. Methods The hemocyanin gene was cloned from hepatopancreas of M.. nipponense using the rapid amplification of cDNA ends (RACE) method,and its sequence was analyzed with a biological software.Spatial and temporal expressions of the gene were detected by real-time PCR. Results The full-length cDNA of hemocyanin was 2 151bp and contained a 14bp of 5ˊ-untranslated region (UTR),a 148bp of 3′UTR and a 189bp of open reading frame (ORF) that encoded a protein of 663 amino acids with a calculated molecular mass of 76.65 kD and pI at 5.42.The deduced amino acid sequence containing three hemocyanin domains and two copper-binding sites exhibited 70% similarity to that of EM>Pacifastacus leniusculus/EM>,63% to that ofEM> Litopenaeus vannamei/EM>.The two copper-binding sites and six histidine residues necessary for the stabilization of the oxygen binding were highly conserved.The 3-D structure modeling of hemocyanin was very similar to that of HC1 from EM>Panulirus interruptus/EM>.Real-time PCR result showed that the gene was expressed highly in the hepatopancreas,weakly in muscles and blood cells,and hardly in the mandibular organ,skin and ovarian.Its transcript derived from hepatopancreas was highest in stage C and lowest in the stage A and D2-3 among the molt cycle.The transcript level of the gene increased significantly 3 hours after injection of the pathogenic bacteria BR>EM>Aeromonas hydrophila/EM>. Conclusion Like other crustaceans,the hemocyanin gene of EM>M.. nipponense/EM> contains the typical hemocyanin domains and conserved copper-binding sites,is highly expressed in hepatopancreas during stage C,and acts as an important molecule involve

Key words: Hemocyanin, Cloning, RACE, Real-time PCR, Spatial and temporal expression, EM>Macrobrachium nipponense/EM>