脊尾白虾血蓝蛋白大亚基基因的克隆及表达分析

应用RACE技术克隆脊尾白虾血蓝蛋白大亚基基因, 并通过攻毒实验揭示脊尾白虾血蓝蛋白基因的先天免疫防御作用, 为脊尾白虾(Exopalaemon carinicauda)的免疫防治研究提供依据和思路。研究成功克隆了脊尾白虾血蓝蛋白大亚基基因全长cDNA序列, 该大亚基cDNA全长 2192 bp, 开放式阅读框长 2034 bp, 5′非编码区长 21 bp, 3′非编码区长 137 bp, 将该基因命名为 EcHcL。EcHcL编码 667 个氨基酸, 前 21 个氨基酸组成信号肽, 推测成熟肽的分子量为 78.5 kD。Blast比对结果显示, 由脊尾白虾血蓝蛋白EcHcL序列推导的氨基酸序列与日本沼虾、凡纳滨对虾血蓝蛋白氨基酸序列的同源性分别达到 87%、73%, 其M结构域氨基酸序列与斑节对虾、日本对虾等物种同源性性高达 90% 左右, 由此推断该cDNA序列属于血蓝蛋白家族。组织表达分析结果显示, EcHcL基因在脊尾白虾鳃、卵巢、肝胰腺、心脏、肠、肌肉、胃、腹神经节、眼柄、血细胞中均有表达, 肝胰腺中相对表达量最高。Real-time PCR分析发现EcHcL基因在金黄色葡萄球菌、副溶血弧菌和对虾白斑综合征病毒(WSSV)感染后脊尾白虾肝胰腺和血细胞中的表达量显著增加, 并具有不同的时空表达模式, 推测脊尾白虾EcHcL基因在免疫防御中具有重要作用。

cDNA CLONING AND EXPRESSION ANALYSIS OF HEMOCYANIN SUBUNIT GENE IN EXOPALAEMON CARINICAUDA

This study cloned the cDNA of hemocyanin subunit from Exopalaemon carinicauda (named EcHcL) by rapid amplification of cDNA ends (RACE). The EcHcL cDNA was 2192 bp in length including a 2034 bp open reading frame (ORF) that encoded a 667 amino acids polypeptide with a signal peptide sequence (1—21 amino acid residues). The predicted molecular weight of the mature peptide was 78.5 kD. The homology analysis revealed that the amino acid sequence of EcHcL was highly conserved with other arthropod. The homology similarities of EcHcL amino acids with Macrobarchium nipponense hemocyanin and Litopenaeus vannamei amino acid sequence were 87% and 73%, respectively. The similarity of the M domain of the amino acid sequence between EcHcL and other shrimp was about 90%. EcHcL was detected in all tested tissues including gill, ovary, hepatopancreas, heart, intestine, muscle, stomach, abdominal ganglion, eyestalk and haemocytes with the highest expression in hepatopancreas. EcHcL was induced distinctly in the hepatopancreas and haemocytes of shrimp by Staphylococcus aureus, Vibrio parahaemolyticus and white spot syndrome virus (WSSV). The results suggest that the different temporal expression pattern of EcHcL might play an important role in shrimp immune system.