作物学报2011，Vol.37Issue(12)：2167-2172,6.DOI:10.3724/SP.J.1006.2011.02167
一种新的快速鉴定蛋白与靶DNA结合位点的方法
A Novel Quick Method for Detecting Target DNA Binding Sites of Protein
摘要
Abstract
DREB (dehydration-responsive element-binding protein) transcription factors play important roles in the stress response and regulation of plants growth and development. In traditional Dnase I Foot printing, DNA probes is labeled with isotope and then performed polyacrylamide gel electrophoresis to separate digested labeled-DNA fragments, which takes various steps, with low differentiation rate and to detect fewer samples. To explore the mechanism of transcriptional regulation of DREB3 in soybean, we used an improved Dnase I Foot-printing method combining with EMSA (electiophoretic mobility shift assay) to identify binding region of proteins and to find out the core element in binding site. In this research, DNA was labeled using fluorescence instead of isotope and automated capillary electrophoresis polyacrylamide gel electrophoresis was replaced to detect digested DNA fragments. Finally, DNA binding site of GmMYB 1 with GmDREB3 promoter was identified rapidly via the modified Dnase I Foot printing. On the other hand, restriction enzyme was used to validate this result. To further confirm binding element in GmDREB3 promoter, we used a putative DNA binding element of GmMYB 1 to complete EMSA, indicating that GmMYB 1 can bind target DNA element in vitro. In short, compared with classic Dnase I Foot printing, the modified method is more rapid, accurate and reliable, which will be advantageous as a high throughout method to largely identification of interaction between protein and target DNA sites in the future.关键词
DNA酶I足迹法/EMSA/毛细管电泳/蛋白与DNA互作Key words
Dnase I foot-printing/Electiophoretic mobility shift assay (EMSA)/Capillary electrophoresis/Interaction between protein and DNA引用本文复制引用
朱明,魏伟,陈明,张宪省,徐兆师,李连城,马有志..一种新的快速鉴定蛋白与靶DNA结合位点的方法[J].作物学报,2011,37(12):2167-2172,6.基金项目
本研究由国家转基因生物新品种培育的重大专项(2008ZX08002-002,2008ZX08002-005)资助. （2008ZX08002-002,2008ZX08002-005）
