2016, 40(6): 856-866. doi: 10.11964/jfc.20151010106
关键词： 魁蚶 , 过氧化氢酶 , RACE , qRT-PCR
Keywords： Scapharca broughtonii , catalase , RACE , qRT-PCR
采用RT-PCR和cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)克隆出魁蚶过氧化氢酶(SbCAT)基因cDNA全长序列,该基因全长为2181 bp,包括1431 bp的开放阅读框(open reading frame,ORF),96 bp的5'端非翻译区(UTR)和654 bp的3'-UTR。其中ORF编码477个氨基酸,预测分子量为54 ku,理论等电点为8.03。SbCAT氨基酸序列与其他所选动物的氨基酸序列具有较高的相似性,其相似度为68%~96%,SbCAT氨基酸具有CAT基因家族的特征性序列,包括CAT活性位点,1个亚铁血红素结合位点及3个催化位点残基。此外,SbCAT还具有保守的亚铁血红素结合口袋与还原型辅酶Ⅱ(NADPH)结合位点。采用实时荧光定量PCR(qRT-PCR)检测了SbCAT的组织表达特征。结果显示,SbCAT mRNA在所检测的6种组织中均有表达,在外套膜中表达量较高,在肝胰腺和红细胞中表达量较低。经鳗弧菌和金黄色葡萄球菌刺激后,鳗弧菌刺激组的外套膜表达量一直较低,其他组织均表现出先上升再下降的趋势。研究表明,SbCAT可能在魁蚶免疫防御中发挥重要作用。
Catalase(EC 1.11.1.6) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to water and oxygen. In the present study, the complete cDNA of Catalase in ark shell Scapharca broughtonii(named SbCAT) was cloned by RT-PCR and rapid amplification of cDNA ends technique, which contained 2181 bp. The cDNA consists of a 5' untranslated region(UTR) of 96 nucleotides, the 3' UTR of 654 bp, and an open reading frame(ORF) of 1431 bp, encoding 477 amino acid residues with 54 ku predicted molecular weight and the theoretical isoelectric point of 8.03. SbCAT amino acid sequence compared with the other animals compared 68%-96%.The deduced amino acid sequence of SbCAT has characteristic features of catalase family such as the catalase active site(61FNRERIPERVV-HAKGAG77), the catalase heme-ligand signature motif(351RLFSYPDTH359) and the three catalytic amino acid residues of His-72, Asn-145 and Tyr-355. In addition, SbCAT also has the conservative heme-binding pocket and NADPH binding sites. Quantitative real-time PCR(qRT-PCR) method was used to analyze the SbCAT mRNA expression characterization in tissues of normal ark shells. The results showed that SbCAT mRNA detected was expressed in six had a high similarity of and it was higher in the mantle and lower in the tissues of hepatopancreas and haemocyte. After Vibrio anguillarum and Staphylococcus aureus challenge, SbCAT expression was rather low in mantle with V.anguillarum challenged, and significantly up-regulated in other tissues. This results suggests that SbCAT may play an important role in the immune defense of S. broughtonii.
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