圆锥小麦ramosa2的克隆及其重组蛋白的DNA结合特性分析  

Cloning of Triticum turgidum L.ramosa2 and DNA Binding Activity Assay of the Recombinant Protein

作　　者：唐如春[1] 杨宇衡[1] 范三红[1,2] 郭蔼光[1,2] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]陕西省农业分子生物学重点实验室,陕西杨凌712100

出　　处：《中国农业科学》2011年第3期439-446,共8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);西北农林科技大学青年学术骨干支持计划项目

摘　　要：【目的】从四倍体圆锥小麦中克隆ramosa2(TtRa2),分析其序列特征、组织表达特异性和体外活性,为阐明ramosa2与麦类作物穗形态建成关系奠定基础。【方法】利用半定量RT-PCR分析ramosa2在不同组织中的表达水平,将其重组到pET-21a-MBP载体并在T7 Express菌株中诱导表达。利用Amylose亲和层析柱纯化重组蛋白,通过凝胶阻滞试验对其DNA特异结合能力进行鉴定。【结果】TtRA2含有LOB和RA2结构域,属于Ⅰ类LBD蛋白家族成员。TtRa2在幼穗中高丰度表达,而在根、茎、叶、种子中未检测到表达。融合的TtRA2主要以可溶性形式存在,其表达量占总蛋白的68.6%。重组TtRA2可与包含核心序列"GCGGCG"的DNA探针特异性结合,两者形成的复合体的解离常数约为10 nmol.L-1。【结论】TtRa2参与圆锥小麦穗形态建成,其编码产物具备RA2-like转录因子的典型特征,具有类似拟南芥LOB的DNA结合特性。【Objective】The ramosa2 gene（TtRa2） was cloned from tetraploid Tritium turgidum L.,and the sequence features,tissue-specific expression pattern and in vitro activity was analyzed,thus providing a foundation for investigating relationship between ramosa2 and spike architecture of Triticeae crops.【Method】Expression profiles of TtRa2 in different tissues were assayed via RT-PCR.TtRa2 was recombined into the vector pET-21a-MBP,and recombinant protein was expressed in E.coli strain T7 Express.MBP fusion protein was purified by amylose affinity chromatography,and its specific DNA binding activity was identified by electrophoretic mobility shift assay（EMSA）.【Result】TtRA2 contains one LOB domain and one RA2 domain,and it belongs to ClassⅠLBD protein family.TtRa2 was highly expressed in spike,while in root,stem,leaf and seed,its transcripts were not detected.Fused TtRA2 mainly existed as soluble form and the percentage of fusion protein was up to 68.6% of total protein.The purified recombinant protein could bind with the DNA probe specifically,which contains the core sequence ＂GCGGCG＂.【Conclusion】TtRa2 involves in the spike morphogenesis process of Triticum turgidum L..It encodes a typical RA2-like transcription factor,which has similar specific DNA binding ability with LOB of Arabidopsis thaliana.

关 键 词：圆锥小麦 ramosa2 原核表达 LOB结构域 凝胶迁移阻滞分析 

分 类 号：S512.1[农业科学—作物学]