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朱鹏, 胡舒, 乔瑞峰, 廖永岩, 王姝懿, 彭金霞, 陆专灵, 韦友传. 卵形鲳鲹组织蛋白酶B基因的克隆及表达分析[J]. 水生生物学报, 2020, 44(2): 289-295. DOI: 10.7541/2020.035
引用本文: 朱鹏, 胡舒, 乔瑞峰, 廖永岩, 王姝懿, 彭金霞, 陆专灵, 韦友传. 卵形鲳鲹组织蛋白酶B基因的克隆及表达分析[J]. 水生生物学报, 2020, 44(2): 289-295. DOI: 10.7541/2020.035
ZHU Peng, HU Shu, QIAO Rui-Feng, LIAO Yong-Yan, WANG Shu-Yi, PENG Jin-Xia, LU Zhuan-Ling, WEI You-Chuan. CLONING AND EXPRESSION ANALYSIS OF CATHEPSIN B GENE OF GOLDEN POMPANO (TRACHINOTUS OVATUS)[J]. ACTA HYDROBIOLOGICA SINICA, 2020, 44(2): 289-295. DOI: 10.7541/2020.035
Citation: ZHU Peng, HU Shu, QIAO Rui-Feng, LIAO Yong-Yan, WANG Shu-Yi, PENG Jin-Xia, LU Zhuan-Ling, WEI You-Chuan. CLONING AND EXPRESSION ANALYSIS OF CATHEPSIN B GENE OF GOLDEN POMPANO (TRACHINOTUS OVATUS)[J]. ACTA HYDROBIOLOGICA SINICA, 2020, 44(2): 289-295. DOI: 10.7541/2020.035

卵形鲳鲹组织蛋白酶B基因的克隆及表达分析

CLONING AND EXPRESSION ANALYSIS OF CATHEPSIN B GENE OF GOLDEN POMPANO (TRACHINOTUS OVATUS)

  • 摘要: 为研究卵形鲳鲹(Trachinotus ovatus)组织蛋白酶B(Cathepsin B, CatB)基因的表达特征和功能, 应用RT-PCR和RACE技术获得卵形鲳鲹CatB(TroCatB)基因的全长cDNA。TroCatB cDNA全长为2181 bp。其中5′UTR和3′UTR分别为391和797 bp, ORF为993 bp, 推定编码330个氨基酸残基, 推定分子质量和理论等电点分别为36.37 kD和5.73。蛋白结构预测TroCatB蛋白具有信号肽(1Met-18Ala)、前体肽(25Leu-64Gly)和一个典型木瓜蛋白酶家族半胱氨酸结构域, 含107Cys、277His、297Asn 3个蛋白酶催化活性位点。同源性分析显示TroCatB蛋白与其他脊椎动物的同源性为67.0%—90.9%, 成熟肽区与其他脊椎动物的同源性为73.7%—92.4%。NJ系统发育树显示卵形鲳鲹和其他鱼类聚为一支, 与髙体鰤距离最近。实时荧光定量PCR检测TroCatB基因mRNA在健康卵形鲳鲹各组织中均有表达, 在脾脏中表达最高; 在溶藻弧菌感染后, TroCatB基因在脾脏、头肾组织中的mRNA表达水平显著上调(P<0.05), 脾脏在感染6h后, 其表达量达到峰值, 头肾则是在12h达到峰值。以上结果表明, TroCatB蛋白的结构域和催化活性位点在遗传进化过程中保守, TroCatB基因参与了机体对细菌免疫的相关生理活动, 在卵形鲳鲹先天性免疫防御中发挥重要作用, 为进一步阐明TroCatB在免疫过程中的功能以及其对病原的抗病机理提供参考。

     

    Abstract: To investigate the expression and function of Trachinotus ovatus cathepsin B (TroCatB) gene in response to bacterial stimulation, TroCatB cDNA was cloned by RT-PCR and RACE technology and it was 2181 bp in length with a 391 bp 5′UTR, a 797 bp 3′UTR and an 993 bp ORF encoding of 330 amino acid residues. The estimated molecular mass and theoretical isoelectric point were 36.37 kD and 5.73, respectively. The TroCatB protein has a signal peptide (1Met-18Ala), a precursor peptide (25Leu-64Gly) and a typical papain family cysteine domain, containing 107Cys, 277His, 297Asn catalytic activity sites. Homology analysis showed that the homology of the TroCatB protein with other vertebrates was 67.0%—90.9%, and the homology of the mature peptide region with other vertebrates was 73.7%—92.4%. The NJ phylogenetic tree revealed that the scorpionfish and other fish clustered together, closest to the corpus callosum. Real-time quantitative PCR indicated that TroCatB mRNA expressed in various tissues with the highest level in spleen. Vibrio alginolyticus infection significantly induced the expression of TroCatB gene in spleen with peak level at 6h and head kidney tissues (P<0.05) with peak level at 12h. These results indicated that the domain and catalytic active sites of TroCatB protein are conserved during genetic evolution. The TroCatB gene regulates the physiological activities of the organism against bacterial immunity, and plays an important role in the innate immune defense of ovate to elucidate the function of TroCatB in the immune process and the pathogenesis of pathogens.

     

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