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作物学报 ›› 2011, Vol. 37 ›› Issue (12): 2167-2172.doi: 10.3724/SP.J.1006.2011.02167

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

一种新的快速鉴定蛋白与靶DNA结合位点的方法

朱明1,2,魏伟2,3,陈明2,*,张宪省1,徐兆师2,李连城2,马有志2   

  1. ZHU Ming1,2,WEI Wei2,3,CHEN Ming2,*,ZHANG Xian-Sheng1,XU Zhao-Shi2,LI Lian-Cheng2,MA You-Zhi2
  • 收稿日期:2011-04-18 修回日期:2011-07-25 出版日期:2011-12-12 网络出版日期:2011-09-29
  • 通讯作者: 陈明, E-mail: chenming@mail.caas.net.cn, Tel: 010-82108789
  • 基金资助:

    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。

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 Published:2011-12-12 Published online:2011-09-29
  • Contact: 陈明, E-mail: chenming@mail.caas.net.cn, Tel: 010-82108789

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摘要： DREB转录因子在植物逆境胁迫响应中起非常重要的作用, 对植物的生长发育起重要的调控作用。传统的DNA酶I足迹法应用同位素标记DNA, 采用序列胶分离DNase I酶切片段, 操作较复杂, 分辨率较低, 不适用于高通量的样品检测。为阐明植物体内DREB转录因子的调控机制, 本研究利用改进的DNA酶I足迹法(DNase I Foot-printing)结合凝胶阻滞法(electrophoretic mobility shift assay, EMSA), 选用荧光色素代替同位素标记, 毛细管电泳技术代替序列胶检测DNase I酶切片段, 判定GmMYB1蛋白与GmDREB3启动子DNA结合的区域。采用限制性内切酶酶切GmDREB3启动子DNA验证以上结果。同时, 在判定的GmMYB1结合区域的基础上, 选择可能的DNA结合元件与GmMYB1进行EMSA试验, 证明GmMYB1可以与相应元件结合。该方法比传统的DNA酶I足迹法快速、简便、准确并可靠, 作为一种高通量的鉴定方法可用于大规模鉴定蛋白质的DNA结合位点。

关键词: DNA酶I足迹法, EMSA, 毛细管电泳, 蛋白与DNA互作

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

[1]Xue L-X(薛丽香), Weng M(翁默), Wu J-F(吴军峰), Zhang Z-Y(张宗玉), Tong T-J(童坦君). Mechanism of transcription regulation mediated by Sp1 and Sp3. Chin J Biochem Mol Biol (中国生物化学与分子生物学报), 2006, 22(2): 106–110 (in Chinese with English abstract)
[2]Verrecchia F, Rossert J, Mauviel A. Blocking sp1 transcription factor broadly inhibits extracellular matrix gene expression in vitro and in vivo: implications for the treatment of tissue fibrosis. J Invest Dermatol, 2001, 116: 755–763
[3]Vergeer W P, Sogo J M, Pretorius P J, de Vries W N. Interaction of Ap1, Ap2, and Sp1 with the regulatory regions of the human pro-alpha1(I)collagen gene. ArchBiochem Biophys, 2000, 377: 69–79
[4]Galas D J, Schmitz A. DNase footprinting: a simple method for the detection of protein-DNA binding specificity. Nucl Acids Res, 1978, 5: 3157–3170
[5]Blanchette M, Schwikowski B, Tompa M. Algorithms for phylogenetic footprinting. J Comput Biol, 2002, 9: 211–223
[6]Rippe R A, Brenner D A, Tugores A. Techniques to measure nucleic acid-protein binding and specificity: nuclear extract preparations, DNase I Footprinting, and mobility shift assays. Methods Mol Biol, 2001, 160: 459–479
[7]Bannister A, Kouzarides T. Basic peptides enhance protein-DNA interaction in vitro. Nucl Acids Res, 1992, 20: 3523
[8]Ochoa A, Brunel F, Mendelzon D, Cohen G N, Zakin M M. Different liver nuclear proteins bind to similar DNA sequences in the 5' flanking regions of three hepatic genes. Nucl Acids Res, 1989, 17: 119–133
[9]Li J J, Herskowitz I. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science, 1993, 262: 1870–1873
[10]Wang C-G(王成刚), Mo Z-H(莫志宏). Progress in methods to study on the interactions between proteins and nucleic acids. Chin Bull Life Sci (生命科学), 2006, 18(2): 195–198 (in Chinese with English abstract)
[11]Galas D J, Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucl Acids Res, 1978, 5: 3157–3170
[12]Bailly C, Kluza J, Martin C, Ellis T, Waring M J. DNase I footprinting of small molecule binding sites on DNA. Methods Mol Biol, 2005, 288: 319–342
[13]Cardew A S, Fox K R. DNase I footprinting. Methods Mol Biol, 2010, 613: 153–172
[14]Hampshire A J, Rusling D A, Broughton-Head V J, Fox K R. Footprinting: a method for determining the sequence selectivity, affinity and kinetics of DNA binding ligands. Methods, 2007, 42: 128–140
[15]Chen M, Xu Z, Xia L, Li L, Cheng X, Dong J, Wang Q, Ma Y. Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene, GmDREB3, in soybean (Glycine max L.). J Exp Bot, 2009, 60: 121–135
[16]Zianni M, Tessanne K, Merighi M, Laguna R, Tabita F R. Identification of the DNA bases of a DNase I footprint by the use of dye primer sequencing on an automated capillary DNA analysis instrument. J Biomolec Tech, 2006, 17: 103–113
[17]Structural Genomics Consortium, China Structural Genomics Consortium, Northeast Structural Genomics Consortium. Protein production and purification. Nat Methods, 2008, 5: 135–146
[18]Connaghan-Jones K D, Moody A D, Bain D L. Quantitative DNase footprint titration: a tool for analyzing the energetics of protein–DNA interactions. Nat Protocols, 2008, 3: 900–914
[19]Gueroult M, Picot D, Abi-Ghanem J, Hartmann B, Baaden M. How cations can assist DNase I in DNA binding and hydrolysis. Plos Comput Biol, 2010, 6: 1–11
[20]Sambrook J, Russell D. Molecular Cloning: A Laboratory Manual. 3rd edition. New York: Cold Spring Harbor Laboratory Press, 2001. pp 1322–1336
[21]Fried M G, Crothers D M. Kinetics and mechanism in the reaction of gene regulatory proteins with DNA. J Mol Biol, 1984, 172: 263–282
[22]Ellis T, Evans D A, Martin C R, Hartley J A. A 96-well DNase I footprinting screen for drug-DNA interactions. Nucl Acids Res, 2007, 35: 1362–4962
[23]Sandaltzopoulos R, Becker P B. Solid-phase DNase I footprinting: quick and versatile. Nucl Acids Res, 1994, 22: 1511–1512
[24]Pan C Q, Ulmer J S, Herzka A, Lazarus R A. Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition, catalysis, and metal ion dependence. Protein Sci, 1998, 7: 628–636
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