目的 阳春砂是中药砂仁的主要来源植物，其转录组中已筛选出预测与萜类合成相关的候选转录因子unigene，对其进行克隆、序列分析和原核表达，以进一步认识阳春砂药效物质萜类合成的调控元件。方法 根据阳春砂转录组数据中预测为AvMYC4b的Unigene0074125序列设计特异引物，提取阳春砂叶片RNA并反转录成cDNA，以此为模板经PCR扩增得到AvMYC4b的核心片段，再通过RACE技术获得全长cDNA，然后进行编码区全长的GATEWAY TOPO克隆。通过LR反应构建原核表达载体，转化大肠杆菌Rosetta（DE3）细胞，以异丙基-β-D-硫代半乳糖苷（IPTG）和阿拉伯糖在16℃下培养诱导融合蛋白表达，收集菌体，经过裂解、超声、纯化，用SDS-PAGE检测蛋白表达结果。结果 克隆获得的AvMYC4b全长有2 579 bp，包含195 bp的5'UTR，1 995 bp的ORF和389 bp的3'UTR，编码664个氨基酸，预测蛋白相对分子质量为72 211。经过生物信息学分析，AvMYC4b氨基酸序列与其他植物的MYC相似，含有转录因子MYC家族的保守结构域，预测可定位于细胞核中。成功构建重组表达载体pDEST17-AvMYC4b并转化Rosetta（DE3）细胞，经诱导表达得到AvMYC4b融合蛋白，SDS-PAGE结果显示蛋白相对分子质量约80 000，与预测相符。结论 从阳春砂中克隆得到bHLH家族转录因子AvMYC4b，并建立了AvMYC4b原核表达系统，为该转录因子的生物功能研究奠定了基础。

Objective The transcription factor AvMYC4b selected from transcriptome databases, which might be closely related to the terpene biosynthesis, was cloned from Amomum villosum for sequence analysis and prokaryotic expression. Methods Based on the transcriptome data of A. villosum, specific primers were designed to obtain the AvMYC4b core sequence. In this study, the whole cDNA sequence of AvMYC4b was obtained by RACE method, then the GATEWAY TOPO cloning vector, and the express vector pDEST17 were constructed by ligation and LR method, respectively, and prokaryotic protein expression was performed. Induced by IPTG and arabinose, the recombinant protein AvMYC4b was successful expressed at the temperature of 16℃. Collected bacteria were processed through lysis, ultrasound and purification, and were used to determine the protein expression by SDS-PAGE. Results AvMYC4b cDNA gene had 2 579 bp, including 165 bp 5'UTR, 1 995 bp ORF, and 389 bp 3'UTR, which encoded a deduce protein of 644 amino acid with a calculated molecular weight of 72 211. Bioinformatics analysis indicated that AvMYC4b had the conserved domain of transcription factor MYC family and predicted that AvMYC4b could be located in nucleus. The SDS-PAGE result showed that the AvMYC4b protein was expressed in Escherichia coil with a molecular mass of about 80 000, which was consistent with the predicted molecular weight. Conclusion AvMYC4b gene of the bHLH family was cloned from A. villosum and had the whole ORF.The recombinant AvMYC4b protein also was successful expressed in Escherichia coil Rosetta (DE3). Therefore, this study could provide fundamental information for the function characterization of AvMYC4b in terpene biosynthesis pathway of A. villosum.

国家自然科学基金青年基金项目（81303163）；广东省高等学校优秀青年教师培养计划（Yq2013042）