鸡RIPK2基因启动子核心区域及生物信息学分析

Core Region of Chicken RIPK2 Gene Promoter and Bioinformatics Analysis

为了对前期高通量测序筛选出的抗禽致病性大肠杆菌鸡中高表达RIPK2基因进行结构和蛋白功能探析,试验通过PCR技术克隆鸡RIPK2起始密码子前3 201 bp的启动子区,构建系列启动子区缺失载体,转染HD11细胞,双荧光素酶试验检测启动子核心区域;5-Azadc、TSA或5-Azadc和TSA联合处理鸡RIPK2启动子;并结合生物信息学技术对鸡RIPK2蛋白的理化性质进行系统分析。结果显示:通过PCR技术成功克隆鸡RIPK2启动子区不同片段,单、双酶切测序结果准确;-2 300~+38 bp区域是鸡RIPK2基因启动子的基本活性区域,-2 300~-1 839 bp之间存在AP-1、CEBPA和NFκB等重要正调控元件。用5-Azadc、TSA以及5-Azadc和TSA联合分别处理鸡RIPK2启动子,均可提高其转录活性。生物信息学分析表明:鸡RIPK2蛋白在细胞核中表达;在生物进化进程中物种间保守性一般,禽类中保守性很高;存在自身磷酸化位点。试验结果为进一步研究鸡RIPK2基因转录调控机制和蛋白功能奠定了基础。

In a previous study,RIPK2 was demonstrated to be highly up-regulated in the chickens resistant to avian pathogenic E.coli,which was screened by high-throughput sequencing.In order to analyze the gene structure and protein function of RIPK2,the promoter containing the 5′-flanking region(3000 bp)and the 3′-flanking region(200 bp)of chicken RIPK2 was cloned by PCR.Then,a serial of truncated promoter fragments were constructed.The vectors were transfected into chicken HD11 cells.The core promoter region was identified by the dual luciferase reporter assay in chicken HD11 cells.5-Azadc,TSA,and the combination of 5-Azadc and TSA were used to treated chicken RIPK2 promoter vector,respectively.The physical and chemical properties of chicken RIPK2 protein were systematically analyzed by bioinformatics technology.The results showed that different fragments of chicken RIPK2 promoter were successfully cloned by PCR,and the results of single and double enzyme digestion and sequencing were accurate.The basic active region of chicken RIPK2 promoter was from-2300 to+38 bp.Also,there were important positive regulatory elements such as AP-1,CEBPA,and NFκB between-2300 to-1839 bp.5-Azadc,TSA,and the combination of 5-Azadc and TSA could improve the transcriptional activity of chicken RIPK2 promoter,respectively.Bioinformatics analysis showed that chicken RIPK2 protein was expressed in the nucleus.In the process of biological evolution,the conservation property of RIPK2 was moderate among species,but high in birds.Chicken RIPK2 protein had autophosphorylation sites.These results lay a theoretical foundation for further study of transcription regulation mechanism and protein function of chicken RIPK2 gene.