Molecular characterization and transcription regulation analysis of type I IFN gene in grass carp (Ctenopharyngodon idella)
Molecular characterization and transcription regulation analysis of type I IFN gene in grass carp (Ctenopharyngodon idella)
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草鱼I型IFN基因的分子特征及转录调控分析
DOI：
10.1016/j.gene.2012.04.091
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发表时间：
2012-08-01
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：
Li, Dongming;Tan, Wanlong;Hu, Chengyu
I型干扰素和干扰素调节因子7 （IRF7）是对病毒感染的先天免疫至关重要的，近年来在许多硬骨鱼中被发现。本研究克隆并鉴定了草鱼I型干扰素（CiIFN）全基因组序列（GU139255）和草鱼IRF7全长cDNA序列（CiIRF7） （GQ141741）。CiIFN由3368 bp组成，保留了鱼类I型ifn特有的5-外显子/4-内含子基因组织。CiIFN横跨5个外显子，编码180个氨基酸的多肽，其中前22个氨基酸代表假定的信号肽。CiIFN启动子序列长度为760 bp，可分为近端区（1 ~ 140 bp）和远端区（400 ~ 700 bp）。CiIRF7的cDNA全长为1808 bp， ORF为1293 bp，编码430个氨基酸。推测的CiIRF7氨基酸序列在n端区域具有dna结合域（DBD）。Real-time PCR分析显示CiIFN在所有组织中均呈低组成表达。经多肌苷：多胞酸（Poly I:C）刺激后，CiIFN在草鱼大部分组织中的表达均显著上调，在脾脏、肾脏和肠道中表达较强。用凝胶迁移位移法分析CiIRF7和CiIRF7- ndbd重组多肽，以及CiIFN启动子序列近端区（CiIFNP2）、远端区（CiIFNP6）和全长区（CiIFNP7）的PCR扩增产物。结果表明，CiIRF7在体外可以结合CiIFN启动子序列的远端区域和近端区域。随后，将CiIFNPs （CiIFNP7/2/6）克隆到pGL3-Basic载体中，将CiIRF7亚克隆到pcDNA3.1载体中，将pGL3-CiIFNPs与pcDNA3.1-CiIRF7分别瞬时转染或共转染到小鼠骨髓瘤细胞系（MMCL） SP2/0和草鱼肾细胞系（CIK）中，通过荧光素酶测定转染细胞中CiIRF7对CiIFN启动子活性的影响。这些结果表明CiIRF7对CiIFN的转录起正向调节作用。(c) 2012 Elsevier B.V.版权所有
Type I interferons and interferon regulatory factor 7 (IRF7), which are crucial for innate immunity against viral infection, have been identified in many teleost fishes in recent years. In this study, the complete genomic sequence of grass carp (Ctenopharyngodon idella) type I interferon (termed CiIFN) (GU139255) and the full-length IRF7 cDNA sequence of grass carp (termed CiIRF7) (GQ141741) were cloned and characterized. CiIFN consists of 3368 bp, retaining the characteristic 5-exon/4-intron gene organization in fish type I IFNs. The CiIFN spans 5 exons and encodes a polypeptide of 180 amino acids, with the first 22 amino acids representing a putative signal peptide. The CiIFN promoter sequence was found to be 760 bp, which can be divided into a proximal region (from 1 to 140 bp) and a distal region (from 400 to 700 bp). The cDNA of CiIRF7 was found to be 1808 bp in full length, with an ORF of 1293 bp that encodes a putative protein of 430 amino acids. The putative amino acid sequence of CiIRF7 possesses a DNA-binding domain (DBD) in the N-terminal region. Real-time PCR analysis revealed that CiIFN displayed a low constitutive expression in all the tissues tested. After stimulation by polyinosinic:polycytidylic acid (Poly I:C), the expression of CiIFN was significantly up-regulated in most tissues of grass carp, with a relatively strong expression in spleen, kidney and intestine. The recombinant polypeptides of CiIRF7 and CiIRF7-nDBD were analyzed in gel mobility shift assays, along with the PCR amplification products of the proximal region (CiIFNP2), the distal region (CiIFNP6) and the full-length (CiIFNP7) of CiIFN promoter sequence. The results revealed that CiIRF7 could bind to the distal region as well as to the proximal region of CiIFN promoter sequence in vitro. Subsequently, the CiIFNPs (CiIFNP7/2/6) were cloned into pGL3-Basic vectors and CiIRF7 was subcloned into pcDNA3.1 vectors, then pGL3-CiIFNPs were separately transiently transfected or co-transfected with pcDNA3.1-CiIRF7 into the mouse myeloma cell lines (MMCL) SP2/0 and the grass carp kidney cell lines (CIK), and the impact of CiIRF7 on CiIFN promoter activity was measured by luciferase assays in transfected cells. These results demonstrated that CiIRF7 acted as a positive regulator on the transcription of CiIFN. (c) 2012 Elsevier B.V. All rights reserved.