日本沼虾血蓝蛋白基因cDNA全长克隆及表达分析

Objective The aim of this study was to clone the hemocyanin gene from Macrobrachium nipponense, and to do the bioinformatics and spatial and temporal expression analysis of the gene. Methods The hemocyanin gene was cloned from hepatopancreas of Macrobrachium nipponense using the rapid amplification of cDNA ends (RACE) method, and its sequence was analyzed with biological software, spatial and temporal expressions of the gene were detected by real-time PCR. Results The full-length cDNA of hemocyanin was 2151bp and contained a 145bp of 5'-untranslated region (UTR), a 14bp of 3'UTR and a 1991bp of open reading frame (ORF) that encoded a protein of 663 amino acids with a calculated molecular mass of 76.65 kDa 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 Pacifastacus leniusculus, 63% to that of Litopenaeus vannamei. 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 Panulirus interruptus. Real-time PCR result showed that the gene was expressed highly in the hepatopancreas, weakly in the 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 D3/D4 among the molt cycle. The transcript level of gene increased significantly 3 h after injection of pathogenic bacteria Aeromonas hydrophila. Conclusion Like other crustaceans, the hemocyanin gene of Macrobrachium nipponense contains the typical hemocyanin domains and conserved copper-binding sites, and is highly expressed in hepatopancreas during stage C, as well as acts as an important molecule involved in immune defence of Aeromonas hydrophila.