鹅MyoG基因启动子活性及转录调控元件分析

doi:10.11843/j.issn.0366-6964.2021.07.010

摘要/Abstract

摘要： 旨在分析鹅MyoG基因启动子活性区域和转录因子，探究该基因的转录调控机制。本研究首先通过PCR扩增泰州鹅MyoG基因5'侧翼区序列1 245 bp并对其进行测序和生物信息学分析，其次，构建4个不同缺失片段的双荧光素酶报告载体，转染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，对转录因子结合位点HNF4（-521～-503 bp）、USF （-379～-370 bp）和E2（-296～-281 bp）进行定点突变并构建突变报告基因载体，在C2C12细胞系内初步鉴定MyoG基因核心转录调控因子。最后，采集70日龄泰州鹅胸肌、腿肌、心、肝、脾、肺、肾和下丘脑组织样，利用荧光定量PCR检测MyoG基因和核心转录调控因子的组织表达谱。结果表明，扩增得到的鹅MyoG基因5'侧翼区序列包含启动子元件；利用双荧光素酶报告载体检测到鹅MyoG基因启动子区-624～-154 bp区域存在关键顺式调控元件；结合定点突变技术初步鉴定USF是鹅MyoG基因核心转录调控元件。组织表达谱研究进一步表明，MyoG和USF基因在鹅8个不同组织中均有表达，且在胸肌、腿肌和心组织中共同高表达（P<0.01）。鹅MyoG基因5'侧翼区具有启动子转录活性，-624～＋37 bp是核心启动子区，USF是MyoG核心转录调控因子。试验结果为探究MyoG基因在鹅肌肉发育过程的调控机制提供理论依据。

关键词: 鹅, MyoG基因, 启动子活性, 点突变, 转录调控

Abstract: The aim of this study was to analyze the activity region and transcription factors in the promoter of goose MyoG gene, and to elucidate the transcriptional regulation mechanism. Firstly, the 1 245 bp 5' flanking region of MyoG was amplified by PCR, followed by sequencing and bioinformatics analysis. Secondly, dual luciferase reporter vectors with 4 different deletion fragments of the promoter were constructed, and then transfected into C2C12 cell lines. Furthermore, the key transcription factor in the core promoter region was predicted using online software, site-directed mutagenesis of the transcription factor binding sites HNF4 (—521-—503 bp), USF (—379-—370 bp) and E2 (—296-—281 bp) were carried out, then 3 mutation reporter vectors were constructed, and the key transcription factors of MyoG gene was preliminary identified in C2C12 cell line. Finally, the expression profile of MyoG and key transcription factor in breast muscle, leg muscle, heart, liver, spleen, lung, kidney and hypothalamus of 70-day-old goose were determined through qPCR. The results showed that the amplified 5' flanking region of MyoG gene contained promoter elements. The key cis-regulatory elements was located at —624-—154 bp region using the dual luciferase reporter vector. Combined with site-directed mutation demonstrated that USF was the key transcription regulatory factor of goose MyoG gene. Tissue expression profile studies further revealed that MyoG and USF expressed in 8 tissues, and they were higher co-expressed in goose breast muscle, leg muscle and heart (P<0.01). The 5' flanking region of goose MyoG had promoter transcriptional activity, the core promoter region was detected at -624-+37 bp, and USF was the key transcription regulatory factor. The experimental results can provide a theoretical basis for exploring molecular regulation mechanism of MyoG gene in goose muscle development.

Key words: goose, MyoG gene, promoter activity, site-directed mutation, transcriptional regulation

中图分类号:

S835.2

陈哲, 陈蓉, 闫乐艳, 陈佳彬, 于建宁. 鹅MyoG基因启动子活性及转录调控元件分析[J]. 畜牧兽医学报, 2021, 52(7): 1869-1879.

CHEN Zhe, CHEN Rong, YAN Leyan, CHEN Jiabin, YU Jianning. Analysis of the Promoter Activity and Transcriptional Regulatory Elements of Goose MyoG Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7): 1869-1879.

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