关键词: 圆锥小麦, ramosa2, 原核表达, LOB结构域, 凝胶迁移阻滞分析

Abstract:

【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.

Key words: Triticum turgidum, ramosa2 gene, prokaryotic expression, LOB domain, electrophoretic mobility shift assay