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周真真, 景斐, 魏可, 张建设. 大黄鱼TRIM25基因克隆和表达分析[J]. 水生生物学报, 2019, 43(6): 1189-1196. DOI: 10.7541/2019.140
引用本文: 周真真, 景斐, 魏可, 张建设. 大黄鱼TRIM25基因克隆和表达分析[J]. 水生生物学报, 2019, 43(6): 1189-1196. DOI: 10.7541/2019.140
ZHOU Zhen-Zhen, JING Fei, WEI Ke, ZHANG Jian-She. MOLECULAR CLONING AND EXPRESSION ANALYSIS OF TRIM25 GENE IN LARIMICHTHYS CROCEA[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(6): 1189-1196. DOI: 10.7541/2019.140
Citation: ZHOU Zhen-Zhen, JING Fei, WEI Ke, ZHANG Jian-She. MOLECULAR CLONING AND EXPRESSION ANALYSIS OF TRIM25 GENE IN LARIMICHTHYS CROCEA[J]. ACTA HYDROBIOLOGICA SINICA, 2019, 43(6): 1189-1196. DOI: 10.7541/2019.140

大黄鱼TRIM25基因克隆和表达分析

MOLECULAR CLONING AND EXPRESSION ANALYSIS OF TRIM25 GENE IN LARIMICHTHYS CROCEA

  • 摘要: 三重基序蛋白25 (Tripartite motif-containing protein 25, TRIM25)属于E3泛素连接酶家族, 在先天免疫反应中发挥重要作用。为研究TRIM25基因在大黄鱼(Larimichthys crocea)先天抗病毒免疫反应中的作用, 研究鉴定并克隆大黄鱼TRIM25基因(命名为LcTRIM25)。LcTRIM25基因编码序列2097 bp (GenBank登录号: MK327541), 编码698个氨基酸。蛋白结构域预测发现LcTRIM25包括保守的RING结构域、B-box2结构域、Coiled-coil结构域和可变的C末端PRY/SPRY结构域。多序列比对以及系统进化树分析表明LcTRIM25基因与斜带石斑鱼同源性高, 与哺乳动物、爬行动物、两栖动物和鸟类同源性相对低, 这说明不同物种受到来自环境不同的选择压力, 导致进化程度不同。应用实时荧光定量PCR方法分析大黄鱼TRIM25基因的表达水平。结果分析发现LcTRIM25基因在健康大黄鱼的9个组织中均有广泛表达, 且在肝脏中表达量最高, 在心脏中表达量最低。在poly(I:C)刺激后, 在外周血、头肾、脾脏和肝脏中LcTRIM25基因表达量迅速且明显上调, 均出现上升达到峰值后下降的趋势。LcTRIM25基因表达量在头肾和脾脏中6h达到最高表达量, 在肝脏中12h达到峰值, 外周血中在24h达到最高表达量。上述结果表明, 不同组织中LcTRIM25基因表达模式具有差异性。研究结果推测大黄鱼TRIM25基因参与抗病毒免疫反应且发挥十分关键的作用, 为进一步了解大黄鱼抗病毒免疫机制提供理论基础。

     

    Abstract: Tripartite motif-containing protein 25 (TRIM25) belongs to the E3 ubiquitin ligase family and plays an important role in innate immune response. To study the role of TRIM25 gene in innate antiviral immune response of large yellow croaker (Larimichthys crocea), the TRIM25 gene (named LcTRIM25) of large yellow croaker was cloned and identified. The coding sequence of LcTRIM25 gene is 2097 bp (GenBank accession numbers: MK327541), encoding 698 amino acid residues. Protein domain analysis found that LcTRIM25 contains a conserved Really Interesting New Gene (RING) domain, a B-box2 domain, a Coiled-coil domain and a variable C-terminal PRY/SPRY domain. Multiple sequence alignment and phylogenetic tree analysis showed that the LcTRIM25 gene had high homology with the Epinephelus coioides, but low homology with mammals, reptiles, amphibians and birds, indicating that the different species were under different selection pressure from environments to reach different evolutionary degrees. The results of real-time fluorescence quantitative PCR revealed that the LcTRIM25 gene was ubiquitously expressed in 9 tested issues of healthy large yellow croaker, with the highest expressions in liver and the lowest expression in heart. The expression of LcTRIM25 gene in peripheral blood, head kidney, spleen and liver were rapidly and significantly upregulated after poly (I:C) stimulation, and then decreased in all tissues. The highest expression level of LcTRIM25 gene was found at 6h in head kidney and spleen, at 12h in liver, and at 24h in peripheral blood, demonstrating that the TRIM25 gene might play a key role in the antiviral immune response of large yellow croaker. This study provides a theoretical basis for further understanding the antiviral immune mechanism of large yellow croaker.

     

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