大黄鱼过氧化氢酶基因的克隆及其对鳗弧菌感染的响应

发布时间：2018-05-03 04:36

本文选题：大黄鱼 + 鳗弧菌　；参考：《水产学报》2017年05期

【摘要】：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。
[Abstract]:In order to investigate the protective effect of catalase (catalase) on large yellow croaker, the complete open reading frame of CAT gene (1584 BP) was obtained based on the genomic sequence database of Pseudosciaena Crocea, which encodes 527 amino acid residues. It contains highly conserved enzyme activity center sequence FDRERIPERVHAKGA, heme hemin binding signal sequence RLFSYPDTH3, three catalytic sites His75 An 148 and Tyr358, and 12 NADPH binding sites. The theoretical molecular weight is 59.98 kuand the isoelectric point is 8.37. Multiple sequence alignment showed that the amino acid sequence of CAT of Pseudosciaena Crocea was highly consistent with other fishes, and had a high homology with the codon and Sparus species belonging to the same family, and was also clustered in the same evolutionary branch in the evolutionary tree, and the amino acid sequence of Pseudosciaena Crocea was similar to that of other fishes. The sequence is a member of CAT family. Real-time fluorescence quantitative PCR analysis showed that CAT gene was expressed in 7 tissues (liver, spleen, brain, kidney, muscle, Gill and intestine), but the highest expression level was found in liver (6.68 times as high as that in muscle). After infection with Vibrio anguilli, the expression of CAT gene in liver tissue of large yellow croaker changed obviously with the passage of time. At 12 hours after infection, the expression of CAT gene reached the highest level of 7.48 times, then decreased gradually, and returned to its original level at 72 h. The expression of cat gene in the control group injected with PBS was only slightly up-regulated, which indicated that the pathogen infection might cause a large number of reactive oxygen free radicals and H _ 2O _ 2 cat gene could scavenge excessive reactive oxygen species in the body and prevent them from damaging the cells.
【作者单位】：浙江海洋大学国家海洋设施养殖工程技术研究中心;
【基金】：国家自然科学基金(41606148) 浙江省自然科学基金(LY14C190004) 浙江省大学生科技创新活动计划暨新苗人才计划(2015R411005) 舟山市科技局项目(2015C41014)~~
【分类号】：S943

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