徐淮山羊c-Myc基因启动子的克隆及其功能的初步分析

doi:10.11843/j.issn.0366-6964.2014.04.004

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (4): 533-540.doi: 10.11843/j.issn.0366-6964.2014.04.004

徐淮山羊c-Myc基因启动子的克隆及其功能的初步分析

韦光辉，左其生，李东，张亚妮，刘志永，朱睿，邱峰龙，李碧春^*

（扬州大学动物科学与技术学院，江苏省动物遗传繁育与分子设计重点实验室，扬州 225009）

收稿日期:2013-11-22 出版日期:2014-04-23 发布日期:2014-04-23
通讯作者: 李碧春，教授，E-mail：yubcli@yzu.edu.cn
作者简介:韦光辉（1982-），男，河南漯河人，博士生，主要从事分子克隆和基因表达调控机制的研究，E-mail：weiguanghui2006@gmail.com
基金资助:
国家转基因重大专项（2013ZX08008-003）

Cloning and Preliminary Function Analysis of Xuhuai Goat c-Myc Promoter

WEI Guang-hui，ZUO Qi-sheng，LI Dong，ZHANG Ya-ni，LIU Zhi-yong，ZHU Rui，QIU Feng-long，LI Bi-chun^*

(Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province，College of Animal Science and Technology，Yangzhou University，Yangzhou 225009， China)

Received:2013-11-22 Online:2014-04-23 Published:2014-04-23

摘要/Abstract

摘要：

本研究旨在确定徐淮山羊c-Myc基因启动子区域，找出该基因启动子的核心调控区，初步探讨c-Myc基因的表达调控机制。根据UCSC基因组数据库已公布的绵羊c-Myc基因的启动子序列，设计特异性PCR引物扩增c-Myc基因的一系列启动子缺失片段，定向克隆至pEGFP-N1和PGL3-c-Myc，分别转染gFF、COS7及P19细胞，并进行TSA和NFAT1诱导，同时对-402～-249 bp区域的转录因子SP1结合位点进行定点突变，最后进行双荧光报告基因活性检测。结果表明，徐淮山羊c-Myc基因5′侧翼区-1 334～+1 bp区域的启动子活性最强，-402～+1 bp区域为c-Myc基因启动子基本活性区域。进一步研究发现，-1 334~-971 bp、-587～-147 bp区域存在正调控元件，-1 976～ -1 334 bp、-971～-587 bp区域存在负调控元件。TSA和NFAT1均能增强c-Myc启动子的活性，SP1结合位点定点突变后，启动子活性降低。本试验通过构建包含c-Myc基因启动子不同片段的重组报告基因载体并比较其转录活性，确定了c-Myc基因启动子的核心区域，发现转录因子SP1是c-Myc基因启动子核心区域的调控元件，为进一步研究c-Myc基因的表达调控机制奠定了基础。

Abstract:

The aim of this study was to determine and find the promoter core regulatory regions of Xuhuai goat’s c-Myc gene，and to investigate c-Myc gene regulation mechanism.According to the sequence of ovis aries c-Myc gene published in GenBank，the primers were designed and the different deletion fragments of 5′ flanking region of c-Myc gene promoter were amplified，and then were cloned into pEGFP-N1 and PGL3-Basic，then were transfected into gFF，COS7 and P19 cells and treated with TSA and NFAT1.SP1 transcription factor binding sites in the region of -402--249 bp was mutated，and the activity of dual-luciferase reporter gene was detected.The study showed that the region containing -1 334-+1 bp of c-Myc promoter had the maximal promoter activities，and sequence of -402-+1 bp had the basal promoter activities，further study showed that there were positive (the region of -1 334--971 bp，-587--147 bp) and negative (the region of -1 976--1 334 bp，-971--587 bp) regulatory domains，respectively.TSA and NFAT1 significantly enhanced the activity of c-Myc promoter.The activity was declined by the point mutation of SP1 binding site.By constructing the recombinant expression vectors containing different fragments of c-Myc gene promoter and comparing their activity，the core regulatory region of c-Myc gene promoter was identified.SP1 is an important regulatory element of c-Myc promoter core region，which lay a foundation for the further research on the mechanism of regulation and expression of c-Myc gene.

中图分类号:

S827
S813.3

韦光辉，左其生，李东，张亚妮，刘志永，朱睿，邱峰龙，李碧春. 徐淮山羊c-Myc基因启动子的克隆及其功能的初步分析[J]. 畜牧兽医学报, 2014, 45(4): 533-540.

WEI Guang-hui，ZUO Qi-sheng，LI Dong，ZHANG Ya-ni，LIU Zhi-yong，ZHU Rui，QIU Feng-long，LI Bi-chun. Cloning and Preliminary Function Analysis of Xuhuai Goat c-Myc Promoter[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(4): 533-540.

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徐淮山羊c-Myc基因启动子的克隆及其功能的初步分析

Cloning and Preliminary Function Analysis of Xuhuai Goat c-Myc Promoter

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