知识与财富的链接
为了探究血蓝蛋白的分子多样性, 对脊尾白虾(Exopalaemon carinicauda)血蓝蛋白大亚基变体进行了研究。基于脊尾白虾转录组文库血蓝蛋白大亚基变体EcHcL1和2序列, 利用RACE和生物信息学技术, 对2种变体进行全长cDNA扩增和序列分析。结果发现EcHcL1和2 cDNA 全长分别为2239和2132 bp, 编码685和676个氨基酸。EcHcL1和2氨基酸序列在进化上相对保守, 且均具有血蓝蛋白的典型结构域, 包括铜离子结合区域、保守His位点和Ig-like区等, 其中EcHcL1多了一个酪氨酸酶结构域, 推测可能与色素代谢及酚氧化酶活性有关。组织表达分布结果显示EcHcL1和2在血细胞、肝胰腺、肠、腹神经节、心脏、卵巢、眼柄、胃、鳃和肌肉等组织中均有表达, 且均在肝胰腺和血细胞中表达较高。采用Real-time PCR方法对病原感染后2种变体的mRNA表达情况进行分析。结果发现, EcHcL1在金黄色葡萄球菌、副溶血弧菌和白斑综合征病毒(WSSV)胁迫脊尾白虾12—48h中出现较高表达量, 最高表达量约为对照组的1.5—4倍; EcHcL2在胁迫后24—48h后, 与对照组相比, 表达量约上升2—9倍。由此说明, 脊尾白虾血蓝蛋白大亚基变体与免疫防御密切相关, 可能在抵抗病原微生物感染中发挥作用。
Two variants of EcHcL1 and 2 based on Exopalaemon carinicauda transcriptome library were obtained by using RACE technique. The total length of EcHcL1 and 2 were 2248 bp and 2121 bp, which encoded protein of 685 and 676 amino acids, respectively. EcHcL1 and 2 are relatively conservative in evolution, and has typical domains of hemocyanin, including copper ion binding region, conserved His site and Ig-like region. EcHcL1 may relate to pigment metabolism and phenoloxidase activity because of more than a tyrosinase domain. The results of tissue expression distribution showed that EcHcL1 and 2 were expressed in all the 10 tissues, and higher expressed in hepatopancreas and haemocytes. Different variants of E. carinicauda hemocyanin subunit may also have various immunological functions. The expression of the 2 variants of mRNA after pathogen stresses were investigated using the Real-time PCR strategy. The results showed that EcHcL1 had higher expression level during 12—48h after Staphylococcus aureus, Vibrio parahaemolyticus and WSSV challenges, and its among more 1.5—4 times higher than that in control group. The expression level of EcHcL2 reached the highest value among 24—48h after stress, which increased 2—9 times compared with the control group. Therefore, we speculated that the E. carinicauda hemocyanin subunit variants may related to immune defenses closely and might have antimicrobial resistance activity.
