中国农业科学2012，Vol.45Issue(1)：7-15,9.DOI:10.3864/j.issn.0578-1752.2012.01.002
小麦WPBF与高分子量谷蛋白基因上游Prolamin-Like box的特异结合
Specific Binding of Wheat WPBF and Prolamin-Like box in Upstream of HMW-GS Genes
摘要
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°C 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.关键词
小麦/WPBF/表达纯化/HMW-GS/Prolamin-Like box/凝胶迁移阻滞试验Key words
wheat/WPBF/expression and purification/HMW-GS/Prolamin-Like box/electrophoretic mobility shift assay引用本文复制引用
卫晓彬,王亚楠,张超,单丽伟,唐如春,范三红..小麦WPBF与高分子量谷蛋白基因上游Prolamin-Like box的特异结合[J].中国农业科学,2012,45(1):7-15,9.基金项目
国家转基因专项"高产转基因小麦新品种选育"(2008ZX08002-003)、中央高校基本科研业务费专项(QN2009070) （2008ZX08002-003）
