伯氏肩孔南极鱼dusp1基因在冷应激中的功能研究

ROLE OF TREMATOMUS BERNACCHII DUSP1 GENE IN CELL COLD RESISTANCE

  • 摘要: 为了研究极地鱼类双特异性磷酸酶1 (dual-specificity phosphatase 1, dusp1)基因在低温胁迫下的作用, 实验采用RT-PCR技术从Trematomus bernacchii中克隆获得了编码区含有1128个核苷酸的dusp1同源基因, 可编码376个氨基酸残基。将其通过同源重组的方法构建真核表达载体pcDNA3.1-dusp1并转染至人胚肾293T(HEK293T)细胞中, 同时以pcDNA3.1空载质粒作为对照。使用荧光探针DCFH-DA 检测了细胞活性氧(Reactive oxygen species assay, ROS)含量, 采用流式细胞术检测了低温胁迫下细胞的存活率, 采用Western Blot检测了P38/MAPK的磷酸化水平和RT-qPCR技术分析了半胱氨酸天冬氨酸蛋白酶3(caspase-3)的mRNA表达水平。结果表明, 伯氏肩孔南极鱼dusp1基因能在293T细胞中大量表达, 并定位于细胞核; 在低温胁迫下, 与对照组相比, 伯氏肩孔南极鱼dusp1基因的过表达能显著减少细胞ROS的含量和细胞凋亡率, 并抑制促凋亡基因P38/MAPK的过度磷酸化和凋亡效应基因caspase-3的上调, 减轻细胞在低温下的受损程度。研究表明伯氏肩孔南极鱼dusp1的过表达提高了293T细胞的抗寒能力, 在细胞低温应激过程中具有保护功能。研究结果为探究极地鱼类低温适应性进化研究提供了新的证据, 同时也为进一步探究冷胁迫下硬骨鱼类 dusp1 的功能奠定了基础。

     

    Abstract: Dual specificity phosphatase 1 (dusp1) is an enzyme to inactivate MAPKs by selectively dephosphorylating critical serine/threonine residues. Dusp1 plays an important role in many environmental stress including hypoxia, oxidative stress, UV and heat shock. However, the role of dusp1 in response to cold stress has been rarely reported in fish species, especially in polar fish. To address this, we cloned a dusp1 homolog gene encoding 376 amino acids from Trematomus bernacchii. The open reading frame encoding dusp1 was amplified by PCR with homologous recombination approach cloned into pcDNA3.1 vector, referred to as pcDNA3.1-dusp1. The constructed eukaryotic expression vector pcDNA3. 1-dusp1 were transferred into the human embryonic kidney 293T (HEK293T) cells, and pcDNA3.1 (empty vector)-transfected cells were used as a negative control. The reactive oxygen species (ROS) was assayed using fluorescence probe DCFH-DA and the survival rate under low temperature stress was detected by flow cytometry, respectively. A Western-blot analysis was applied to determine the phosphorylation of P38/MAPK levels and the apoptosis effector gene caspase-3 mRNA expression levels were measured using RT-qPCR. The results showed the T. bernacchii DUSP1 protein in the HEK293T cells with the subcellular localization at nucleus. Dusp1 over-expression significantly decreased ROS content and apoptosis rate in cells under cold stress, and it also suppressed hyperphosphorylation of the pro-apoptotic gene p38 and decreased caspase-3 transcriptional activity. These results indicated that dusp1 from T. bernacchii can protect cells from the damage under cold stress, and this protection was achieved by inhibiting of the p38MAPK and caspase-3 activation. This study provide new insights of the cold temperature adaptive evolution of polar fish and lay a foundation for further functional studies of dusp1 in teleosts under cold stress.

     

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