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目的克隆日本沼虾血蓝蛋白基因,进行生物信息学及时空表达分析。方法利用cDNA末端快速扩增技术(RACE)从肝胰腺中克隆血蓝蛋白基因cDNA全长序列,用生物学软件对其序列进行生物信息学分析,时空表达分析采用实时荧光定量PCR方法。结果日本沼虾血蓝蛋白基因cDNA全长2 151 bp,包含14 bp的5’UTR、148 bp的3’UTR和189 bp的开放阅读框(ORF)。ORF编码663个氨基酸,预测分子量为76.65kD,理论等电点为5.42,含有典型的3个血蓝蛋白结构域和2个保守的铜离子结合位点。与淡水螯虾、凡纳滨对虾血蓝蛋白相似性分别为70%和63%。该基因在肝胰腺中的相对表达量最高,肌肉和血细胞表达较弱,大颚器官、表皮和卵巢几乎不表达;肝胰腺血蓝蛋白基因的表达量在蜕皮间期最高,蜕皮后期和蜕皮前期较低;嗜水气单胞菌刺激后肝胰腺中的表达量显著增加。结论与其他甲壳动物相似,日本沼虾血蓝蛋白基因具有典型的结构域和保守的铜离子结合位点,在蜕皮间期的肝胰腺中呈高表达,是参与机体免疫防御反应的一种重要分子。
Objective To clone the hemocyanin gene of Macrobrachium nipponense for bioinformatics and spatiotemporal expression analysis. Methods The full-length cDNA of hemocyanin gene was cloned from hepatopancreas by rapid amplification of cDNA ends (RACE). Bioinformatic analysis of its sequence was performed by using biological software. Real-time PCR was used to analyze the spatiotemporal expression. Results The cDNA of hemocyanin from M. japonicus was 2 151 bp in length, including 14 bp 5’UTR, 148 bp 3 ’UTR and 189 bp open reading frame (ORF). The ORF encoded 663 amino acids with a predicted molecular weight of 76.65 kD and a theoretical isoelectric point of 5.42. It contained three typical hemocyanin domains and two conserved copper ion binding sites. Similarities to freshwater crayfish and shrimp Litopenaeus vannamei were 70% and 63%, respectively. The gene was expressed most abundantly in hepatopancreas, but weakly in muscle and blood cells, and hardly expressed in the organs, epidermis and ovary. The expression of hepatopancreas protein was the highest in the intermolecular interphase, late eclampsia and pre-eclampsia Lower; the amount of expression in hepatopancreas significantly increased after Aeromonas hydrophila stimulation. Conclusion Similar to other crustaceans, the MHC gene of M. japonicus has a typical domain and a conserved copper ion binding site, which is highly expressed in the hepatomegaly of the ecdysteric interphalacosis and is an important factor involved in the immune defense response in the body molecular.