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

作　　者：卫晓彬[1] 王亚楠[1] 张超[1] 单丽伟[1] 唐如春[1] 范三红[1] 

机构地区：[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

出　　处：《中国农业科学》2012年第1期7-15,共9页Scientia Agricultura Sinica

基　　金：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)

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.coliT7 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。[ 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.

关 键 词：小麦 WPBF 表达纯化 HMW-GS Prolamin—Like BOX 凝胶迁移阻滞试验 

分 类 号：S512.1[农业科学—作物学]