小麦WPBF与高分子量谷蛋白基因上游Prolamin-Like box的特异结合

doi:10.3864/j.issn.0578-1752.2012.01.002

摘要/Abstract

摘要： 【目的】克隆普通小麦胚乳特异表达转录因子基因WPBF，对其原核表达条件进行优化，并验证其编码蛋白和小麦高分子量谷蛋白亚基基因（HMW-GS）上游Prolamin-Like box的结合特性，为小麦HMW-GS表达调控机制的阐明奠定基础。【方法】利用RT-PCR扩增得到WPBF编码区，将其连接到pET-21a-MBP载体中，并分别在E.coli T7 Express和Origami B（DE3）菌株中诱导表达。依次利用Amylose和Ni-NTA亲和层析柱纯化重组蛋白，最后通过凝胶迁移阻滞试验（EMSA）对重组蛋白的DNA结合特性进行分析。【结果】在33℃的Origami B（DE3）菌株中表达含MBP和His双标签的重组WPBF大部分以可溶性形式存在，其表达量占细菌总蛋白的48.0%。重组WPBF和含HMW-GS上游Prolamin-Like box的两类DNA探针能发生特异结合，但重组WPBF和含“TGCAAAG”基序DNA探针的结合强度高于其和含“TGCAAG”基序DNA探针的结合。【结论】建立了优化的WPBF原核表达系统，并获得具有生物活性的重组蛋白；重组WPBF能与HMW-GS启动子来源的两种Prolamin-Like box特异性结合, 但结合能力存在差异。

关键词: 小麦, WPBF, 表达纯化, HMW-GS, Prolamin-Like box, 凝胶迁移阻滞试验

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

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.

WEI Xiao-Bin, WANG Ya-Nan, ZHANG Chao, DAN Li-Wei, TANG Ru-Chun, FAN San-Hong. Specific Binding of Wheat WPBF and Prolamin-Likebox in Upstream of HMW-GS Genes[J]. Scientia Agricultura Sinica, 2012, 45(1): 7-15.

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