As a key enzyme in the antioxidant systems of living organisms, catalase plays an important role in eliminating hydrogen peroxide against oxidative stress. The open reading frame (ORF) of catalase (GenBank Accession No. KKF14425.1) was cloned from large yellow croaker (Larimichthys crocea), which comprised 1584 bp, encoding a peptide of 527 amino acids (aa) in length, with a predicted molecular mass of 59.98 kDa and a theoretical isoelectric point of 8.37. Several highly conserved motifs, including the proximal active site signature"FDRERIPERVVHAKGA", the proximal heme-ligand signature sequence "RLFSYPDTH", the three catalytic amino acid residues of His75, Asn148 and Tyr358 and NADPH binding site were identified in the deduced amino acid sequence ofCAT from large yellow croaker. Sequence comparison strongly suggested that this sequence was a member of CAT family and highly homologous with other known CAT of fish, especialy with Rachycentron canadum and Oplegnathus fasciatus (94%) of Sciaenidae, and they also gathered into the same branch in the phylogenetic tree. Constitutive CAT mRNA expression was detected in seven tissues of large yellow croaker with different magnitudes, which was high in liver and low in muscle, suggesting its diverse role in physiology with respect to the tissue type. The mRNA of HSP70 in liver after infection by Vibrio anguillarum was detected based on RT-PCR analysis. The transcriptions of CAT were upregulated, the maximum level appeared at 12 h post-injection with 7.48-fold and dropped back to the original level at 72 h post-injection, which showed a slight rise in the group of PBS injection. The results indicated that CAT inL. crocea can function as a potent antioxidant enzyme in large yellow croaker and may play a role in postimmune responses with respect to its peroxidase activity.%为探究过氧化氢酶(catalase,CAT)对大黄鱼机体的保护作用,实验基于大黄鱼基因组序列数据库克隆获得CAT基因1584 bp的完整开放阅读框(GenBank登陆号:KKF-14425.1),该序列编码527个氨基酸残基,包含与其他动物高度保守的酶活性中心序列FDRERIPERVVHAKGA、亚铁血红素结合信号序列RLFSYPDTH、3个催化位点残基His75、Asn148和Tyr358,以及12个NADPH结合位点等,理论分子量为59.98 ku,等电点为8.37.多序列比对显示,大黄鱼CAT氨基酸序列与其他鱼类具有较高的一致性,与同属于石首鱼科的军曹鱼和条石鲷同源性高达94%,在进化树中也聚类于同一进化分支,说明该序列为CAT家族成员.实时荧光定量PCR检测显示,大黄鱼CAT基因在所检测的7种组织(肝脏、脾脏、脑、肾、肌肉、鳃、肠)中均有表达,但在肝脏中表达水平最高(为肌肉中的6.68倍).鳗弧菌感染后,大黄鱼肝组织中CAT基因的表达随着时间的推移而变化明显,感染后12 h,达到最高(7.48倍),随后逐渐下降,到72 h已基本恢复到原始水平,注射PBS的对照组,CAT基因表达只略有上调,说明病原菌侵染可能引起鱼体产生大量活性氧自由基及H2O2,CAT基因则可以清除体内过量的活性氧,进而防止它们对细胞造成损伤.
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