Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (1): 7-15.doi: 10.3864/j.issn.0578-1752.2012.01.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Specific Binding of Wheat WPBF and Prolamin-Likebox in Upstream of HMW-GS Genes

 WEI  Xiao-Bin, WANG  Ya-Nan, ZHANG  Chao, DAN  Li-Wei, TANG  Ru-Chun, FAN  San-Hong   

  1. 1.西北农林科技大学生命科学学院/旱区作物逆境生物学国家重点实验室，陕西杨凌 712100
  • Received:2011-07-06 Online:2012-01-01 Published:2011-10-17

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Abstract: 【Objective】 The endosperm-specific transcription factor gene WPBF was cloned from wheat (Triticum aestivum L.), an optimized prokaryotic expression system was established, and the specific binding between WPBF and Prolamin-Like boxes in upstream of HMW-GS genes was verified. These will be useful for further elucidating the regulation mechanism of HMW-GS. 【Method】 The coding region of WPBF was obtained by RT-PCR and inserted into the vector pET-21a-MBP. The recombinant vector was expressed in E. coli strains T7 Express and Origami B(DE3), the recombinant protein was purified by amylose and Ni-NTA affinity chromatography, and its DNA binding activity was analyzed by electrophoretic mobility shift assay (EMSA).【Result】 Recombinant WPBF existed mainly in soluble form when it was expressed at 33℃ in Origami B (DE3) strain, and the fusion protein accounted for 48% of the total cell protein. The purified recombinant protein could bind specifically to two kinds of Prolamin-Like boxes，however，the binding strength between WPBF and Prolamin-Like box “TGCAAAG” was higher than WPBF and Prolamin-Like box“TGCAAG”. 【Conclusion】 An optimized prokaryotic expression system was established and the functional recombinant protein was obtained. The recombinant WPBF can bind specifically to two kinds of Prolamin-Like boxes in upstream of HMW-GS, and there is somewhat different in binding strength.

Key words: wheat, WPBF, expression and purification, HMW-GS, Prolamin-Like box, electrophoretic mobility shift assay

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