摘要
旨在确定Piwil1基因核心启动子活性调控区,预测转录因子结合位点,并探讨转录因子对家禽Piwil1基因核心启动子活性的影响。通过将Piwil1基因启动子系列缺失片段插入到pGL3-basic载体,构建重组体质粒,将重组体分别转染COS-7和GC-1细胞系,使用双荧光素酶报告基因检测系统测定其转录活性,确定Piwil1基因核心启动子活性调控区,并对调控区进行转录因子结合位点预测;对预测的转录因子NF-Y结合位点进行定点突变,构建重组体质粒,并通过双荧光素酶报告基因检测系统分析其对启动子活性的影响。狼山鸡Piwil1基因的-1 377^-268bp区域在COS-7和GC-1细胞中都有不同程度的启动活性,-221^+1bp启动子活性几乎完全丧失,在-268^-221bp区域有转录因子NF-Y结合位点,定点突变后启动子活性显著下降,认为转录因子NF-Y对启动子活性可能起着重要的调控作用。转录因子NF-Y为Piwil1基因核心启动子区的重要调控元件,为进一步研究Piwil1基因的转录调控机制奠定了理论基础。
The aim of this study was to identify the active control area of the chicken Piwil1 gene core promoter, to predict the binding sites of the transcription factor, and to investigate the effect of the NF-Y binding sites on the chicken Piwil1 gene core promoter activity. A series of promot- er deletion mutants were directly subcloned into pGL3-Basic vector. The transcriptional activity of the recombinant plasmids was detected by Dual-Glo Luciferase Assay System after transfect- ing COS-7 and GC-1 ceils. In the deletion analysis of Piwil1 promoter, the core promoter essen- tial for transcriptional activation was identified. With point mutations and Dual-Glo Luciferase Assay System,the effect of the NF-Y binding site on the chicken Piwil1 gene core promoter ac- tivity were identified. The activity of the --1 377-268 bp region in COS-7 and GC-1 ceils were different, while the activity of --221-＋1 bp region was almost disappeared. Data analysis demon- strated that there was the NF-Y binding site in --268-221 bp region. By point mutations, the activity was decreased significantly. Therefore, NF-Y site might has an important effect on Pi- will gene promoter activity. NF-Y is an important regulatory element in Piwil1 core promoter. Furthermore, the results provide the foundation for analyzing the promoter activity and transcrip-tional regulation mechanism.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2013年第9期1349-1354,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(31172199)
国家科技支撑计划(2011BAD28B03)
国家高技术研究发展计划(863计划)项目(2011AA100305)
作者简介
夏明秀（1987-），女，山东潍坊人，硕士，主要从事家禽遗传育种研究，E—mail：Xmingxiuia@126．com.
通信作者：常国斌，副教授，E-mail：passioncgb@163．com；
通信作者：陈国宏，教授，Email：ghchen@yzu．edu．cn.
