title: The transcription regulation analysis of Ctenopharyngodon idellus PKR and PKZ genes.
abstract: 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.
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Liu
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Huiling
authors.initials: H
authors.lastname: Mao
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Meihui
authors.initials: M
authors.lastname: Gu
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Xu
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Zhicheng
authors.initials: Z
authors.lastname: Sun
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Lin
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Haizhou
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Dingkun
authors.initials: D
authors.lastname: Xie
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Qunhao
authors.initials: Q
authors.lastname: Hou
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Xiangqin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Yichuan
authors.initials: Y
authors.lastname: Mi
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China.
authors.forename: Xiancheng
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang 330031, China. Electronic address: hucy2008@163.com.
authors.forename: Chengyu
authors.initials: C
authors.lastname: Hu
mesh_terms: D000818:Animals; D002347:Carps / Q000235:genetics*; D005786:Gene Expression Regulation; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D011494:Protein Kinases
keywords: IRF3 and IRF7; ISG; ISRE; PKR and PKZ; Transcriptional regulation