牛IRX3基因启动子转录调控分析

doi:10.11843/j.issn.0366-6964.2020.05.011

摘要/Abstract

摘要： 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。

关键词: IRX3基因, 启动子, 转录调控

Abstract: The aim of this study was to elucidate the transcriptional regulation mechanism and expression patterns of bovine IRX3 gene and identify the key transcription factors of promoter. The distribution of bovine IRX3 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 IRX3 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 IRX3 gene was identified in 3T3-L1 cell line by site-directed mutation and siRNA interference. The results showed that IRX3 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 IRX3 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 IRX3 gene. The tissue expression analysis results indicated that the expression level of IRX3 was relatively higher in longissimus dorsi and subcutaneous fat. There were 8 CpG islands in the 1.8 kb region of IRX3 promoter. NRF1, KLF4, HOXA5 and CREB1 were located in CpG islands and regulated the transcriptional activity of IRX3 gene. These results lay an important theoretical foundation for exploring the molecular regulation mechanism of IRX3 gene in bovine fat deposition.

Key words: IRX3 gene, promoter, transcriptional regulation

中图分类号:

S823.2

魏大为, 杨智燕, 王兴平, 罗仍卓么, 张久盘. 牛IRX3基因启动子转录调控分析[J]. 畜牧兽医学报, 2020, 51(5): 997-1006.

WEI Dawei, YANG Zhiyan, WANG Xingping, LUO Rengzhuoma, ZHANG Jiupan. Transcription Regulation Analysis of Bovine IRX3 Gene Promoter[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 997-1006.

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