脊尾白虾(Exopalaemon carinicauda) 2-巨球蛋白cDNA全长的克隆和表达分析

The Cloning and Expression of Alpha2-Macrogloblin Gene of Exopalaemon carinicauda

中文关键词: 脊尾白虾  α2-巨球蛋白  基因克隆  基因表达

英文关键词: Exopalaemon carinicauda  alpha2-macrogloblin  Gene cloning  Gene expression

中文摘要:

      根据本实验室前期获得的脊尾白虾(Exopalaemon carinicauda) α2-巨球蛋白基因EST序列，采用cDNA末端快速扩增(Rapid amplification of cDNA end, RACE)技术克隆获得脊尾白虾α2-巨球蛋白基因cDNA全长，命名为Ecα2M基因。该基因全长4823 bp，由4413 bp的开放阅读框、64 bp的5端非编码区以及346 bp的3端非编码区组成。开放阅读框编码1470个氨基酸，分子量为163.0 kDa，理论等电点为5.03。序列分析显示，Ecα2M序列N端含有23个氨基酸组成的信号肽。同源性分析显示，脊尾白虾Ecα2M氨基酸序列与罗氏沼虾(Macrobrachium rosenbergii) α2M的同源性最高，达到80%。荧光定量PCR分析结果显示，Ecα2M基因在血细胞、肝胰腺、肌肉、鳃、卵巢、眼柄、胃及肠中均有表达，其中在血细胞中的相对表达量最高。感染鳗弧菌和WSSV后，脊尾白虾血细胞中Ecα2M的相对表达量于6 h达到最大值且显著高于对照组(P<0.05)，肝胰腺中Ecα2M的相对表达量于3 h达到最大值且显著高于对照组(P<0.05)，相对表达量变化具有明显的时间差异性。

英文摘要:

      Exopalaemon carinicauda is one of most commercially important shrimps in China Sea due to its advantage in reproduction, longer growing seasons, and ecological flexibility. However, a variety of shrimp diseases have become an increasingly serious issue that lead to economic loss. E. carinicauda relies on the innate immunity to fight against invading pathogens. Alpha2-Macrogloblin (α2M) is an inhibitor of diverse proteases ubiquitous in animals. Alpha2M enfolds the target proteases and block their interactions. Therefore α2M plays an important part in avoiding the deleterious effects of its active components. Based on the EST sequence from a cDNA library of E. carinicauda hemocyte，we cloned the cDNA of alpha2-macrogloblin of E. carinicauda (namely Ecα2M) using rapid amplification of cDNA ends (RACE). The Ecα2M cDNA was 4823 bp in length, which contained an open reading frame (ORF) of 4413 bp, a 64 bp 5 untranslated region, and a 346 bp 3 untranslated region. The open reading frame encoded an N-terminal signal sequence of 23 residues, and a mature protein consisting of 1470 amino acids. The molecular mass of this protein was 163.0 kDa and the estimated pI was 5.03. Homology analysis revealed that the amino acid sequence of Ecα2M was highly similar to its homologs in other crustaceans. The similarity of Ecα2M was 80% between E. carinicauda and Macrobrachium rosenbergii. We also analyzed the expression of Ecα2M in different tissues with quantitative real-time PCR. Ecα2M was detected in all tested tissues of E. carinicauda, including hemocytes, gill, hepatopancreas, muscles, ovary, intestine, stomach and eyestalk, and the expression was the highest in hemocytes. After challenged with Vibrio anguillarum and white spot syndrome virus (WSSV), the expression of Ecα2M was up-regulated in hemocytes and hepatopancreas of E. carinicauda. Our results suggested that Ecα2M might play an important role in the prawn immune response.