牛IRX3基因启动子转录调控分析

Transcription Regulation Analysis of Bovine IRX 3 Gene Promoter

旨在探究牛IRX3基因组织表达规律,并鉴定其启动子区关键转录因子,以期阐明其转录调控机制。本研究采集3头成年公牛心、肝、脾、肺、肾、皮下脂肪、背最长肌、大肠、小肠、网胃、瘤胃、皱胃及睾丸组织,利用荧光定量PCR检测IRX3基因在不同组织中相对表达量。同时克隆牛IRX3基因1.8 kb启动子区序列并构建其启动子6个不同缺失片段的双荧光素酶报告载体,分别转染3T3-L1和C2C12细胞系。进一步利用在线软件预测核心启动子区关键转录因子,借助定点突变及siRNA干扰技术在3T3-L1细胞系内初步鉴定关键转录因子对IRX3基因的转录调控作用。结果表明,IRX3基因在牛13个不同组织中均有表达,且在肺、肾、心、皮下脂肪、背最长肌中高表达(P<0.05)。利用双荧光素酶报告载体检测到牛IRX3基因核心区域在-372/-42 bp。结合定点突变及siRNA干扰技术初步鉴定NRF1、KLF4、HOXA5和CREB1转录因子对IRX3基因的转录活性有重要的调控作用。综上表明,牛IRX3基因在背最长肌和脂肪组织中表达量相对较高,其启动子1.8 kb区有8个CpG岛,核心区关键转录因子NRF1、KLF4、HOXA5和CREB1位于CpG岛内且调控IRX3基因转录活性。本研究结果为探究IRX3基因在牛脂肪沉积过程中的分子调控机制奠定了重要理论基础。

The aim of this study was to elucidate the transcriptional regulation mechanism and expression patterns of bovine IRX 3 gene and identify the key transcription factors of promoter.The distribution of bovine IRX 3 mRNA in 3 adult bulls tissues was determined through qPCR,including heart,liver,spleen,lung,kidney,subcutaneous fat,longissimus dorsi,large intestine,small intestine,reticulum,rumen,abdomen and testicle.In addition,the 1.8 kb promoter sequence of IRX 3 gene was cloned and dual luciferase receptor vectors with the 6 different deletion fragments of the promoter were constructed,and then transfected into 3T3-L1 and C2C12 cell lines,respectively.Furthermore,the key transcription factors in the core promoter region were predicted by online software,and the transcriptional regulation of key transcription factors on IRX 3 gene was identified in 3T3-L1 cell line by site-directed mutation and siRNA interference.The results showed that IRX 3 had a broad tissue distribution in 13 tissues,and it was highly expressed in the lung,kidney,heart,subcutaneous fat and longissimus dorsi(P<0.05).The promoter core region of bovine IRX 3 gene was located at-372/-42 bp using the dual luciferase reporter vector.Combined with site-directed mutation and siRNA interference demonstrated that NRF1,KLF4,HOXA5 and CREB1 regulated the transcriptional activity of IRX 3 gene.The tissue expression analysis results indicated that the expression level of IRX 3 was relatively higher in longissimus dorsi and subcutaneous fat.There were 8 CpG islands in the 1.8 kb region of IRX 3 promoter.NRF1,KLF4,HOXA5 and CREB1 were located in CpG islands and regulated the transcriptional activity of IRX 3 gene.These results lay an important theoretical foundation for exploring the molecular regulation mechanism of IRX 3 gene in bovine fat deposition.