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Acta Agron Sin ›› 2011, Vol. 37 ›› Issue (12): 2167-2172.doi: 10.3724/SP.J.1006.2011.02167


A Novel Quick Method for Detecting Target DNA Binding Sites of Protein

1 山东农业大学生命科学学院, 山东泰安 271018; 2农作物基因资源与基因改良国家重大科学工程 / 农业部作物遗传育种重点开放实验室 / 中国农业科学院作物科学研究所, 北京 100081; 3 内蒙古农业大学农学院, 内蒙古呼和浩特 010018   

  1. 1 College of Life Sciences, Shandong Agricultural University, Tai’an 271018, China; 2 National Key Facility for Crop Gene Resources and Genetic Improvement / Key Laboratory of Crop Genetics and Breeding of Ministry of Agriculture / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 3 Agriculture College, Inner Mongolia Agricultural University, Hohhot 010018, China
  • Received:2011-04-18 Revised:2011-07-25 Online:2011-12-12 Published:2011-09-29
  • Contact: 陈明, E-mail: chenming@mail.caas.net.cn, Tel: 010-82108789

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 oftranscriptional regulation of DREB3 in soybean, we used an improved DNase I Foot-printing method combining with EMSA (electrophoretic 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 GmMYB1 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 GmMYB1 to complete EMSA, indicating that GmMYB1 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.

Key words: DNase I foot-printing, Electrophoretic mobility shift assay (EMSA), Capillary electrophoresis, Interaction between protein and DNA

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