The transcription regulation analysis of Ctenopharyngodon idellus PKR and PKZ genes
The transcription regulation analysis of Ctenopharyngodon idellus PKR and PKZ genes
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草鱼PKR和PKZ基因转录调控分析
DOI：
10.1016/j.gene.2015.10.070
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发表时间：
2016-01-15
期刊：
影响因子：
3.5
通讯作者：
Hu, Chengyu
中科院分区：
文献类型：
--
作者：
Liu, Dan;Mao, Huiling;Hu, Chengyu
蛋白激酶 R (PKR) 是一种双链 RNA 激活蛋白激酶，存在于哺乳动物和鱼类中。 PKZ 是一种包含 Z-DNA 结合域的 PKR 样蛋白激酶，仅存在于鱼类中。 PKR 和 PKZ 通过抑制细胞内蛋白质翻译在抗病毒防御中发挥协同作用。转录因子 IRF3（干扰素调节因子 3）是 I 型 IFN（干扰素）和 ISG（干扰素刺激基因）的关键调节因子。在之前克隆的CiIRF3的基础上，在BL21大肠杆菌中过表达带His标签的CiIRF3，并用Ni-NTA His-Bind Resin亲和层析纯化表达的蛋白。在本研究中，我们证明了草鱼 (Ctenopharyngodon idellus) PKR (CiPKR) 和 PKZ (CiPKZ) 基因可以在草鱼肾 (CIK) 细胞中被 Poly I:C 诱导。因此，它们可能与细胞内抗病毒活性有关。为了了解病毒诱导后CiPKR和CiPKZ基因的上调机制，我们根据CiPKR（KJ704845）的启动子序列构建了野生型（pGL3-CiPKR-luc和pGL3-CiPKZ-luc）和突变体（pGL3-CiPKR-nISRE-luc和pGL3-CiPKZ-nISRE-luc）报告基因载体 和 CiPKZ (KJ704844)。体外凝胶迁移率变化分析表明，CiIRF3 能够以高亲和力结合 CiPKR 和 CiPKZ 启动子。然而，CiIRF3 与突变体 CiPKR-nISRE 和 CiPKZ-nISRE 的结合微弱。随后将重组质粒pGL3-CiPKR-luc、pGL3-CiPKZ-luc分别与pcDNA3.1-CiIRF3、pcDNA3.1-CiIRF7瞬时共转染CIK细胞。细胞转染实验表明 CiIRF3 和 CiIRF7 上调 CiPKR 和 CiPKZ 的转录水平。结果还表明，ISRE（干扰素刺激反应元件）的共有序列是CiPKR和CiPKZ转录起始的重要调控元件。 (c) 2015 Elsevier B.V. 保留所有权利。
Protein kinase R (PKR), the double-stranded RNA-activated protein kinase, exists in mammalian and fish. PKZ, a PKR-like protein kinase containing Z-DNA binding domains, just exists in fish. PKR and PKZ work synergistically in the antiviral defense by inhibiting intracellular protein translation. The transcriptional factor IRF3 (interferon regulatory factor 3) acts as a key regulator of type I IFN (Interferon) and ISG (interferon stimulated gene). On the basis of the cloned CiIRF3 previously, CiIRF3 with His-tag was over-expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin. In this study, we have demonstrated that grass carp (Ctenopharyngodon idellus) PKR (CiPKR) and PKZ (CiPKZ) genes were inducible by Poly I:C in C. idella kidney (CIK) cells. So, they might be implicated in the intracellular antiviral activity. To understand the up regulatory mechanism of CiPKR and CiPKZ genes upon virus induction, we constructed wild type (pGL3-CiPKR-luc and pGL3-CiPKZ-luc) and the mutant (pGL3-CiPKR-nISRE-luc and pGL3-CiPKZ-nISRE-luc) reporter gene vectors according to the promoter sequences of CiPKR (KJ704845) and CiPKZ (KJ704844). In vitro, gel mobility shift assays demonstrated that CiIRF3 can combine CiPKR and CiPKZ promoters with high affinity. However, CiIRF3 bound to the mutants CiPKR-nISRE and CiPKZ-nISRE faintly. Whereafter, the recombinant plasmids of pGL3-CiPKR-luc, pGL3-CiPKZ-luc were transiently co-transfected with pcDNA3.1-CiIRF3, pcDNA3.1-CiIRF7 respectively into CIK cells. Cell transfection assays indicated that CiIRF3 and CiIRF7 up-regulated the transcriptional level of CiPKR and CiPKZ. The results also revealed that the consensus sequence of ISRE (interferon stimulated response element) is an important regulatory element for the transcriptional initiation of CiPKR and CiPKZ. (c) 2015 Elsevier B.V. All rights reserved.