项目名称： 枯草芽孢杆菌稳定期高效表达启动子P16功能元件的解析及优化

项目编号： No.31500064

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 刘晓青

作者单位： 中国农业科学院生物技术研究所

项目金额： 18万元

中文摘要： 枯草芽孢杆菌是重要的外源基因表达宿主，强而可控的启动子是其应用的关键因素。特异在稳定期高效表达目的基因的自诱导型启动子，不需添加诱导物即可高效表达目的基因，是基因表达系统中的理想启动子。通过对枯草芽孢杆菌启动子相关元件的解析及优化，可解释其在稳定期特异高效表达目的基因的分子机制，具有重要的理论意义及应用前景。我们前期以枯草芽孢杆菌在不同生长时期的转录谱数据为基础，筛选得到稳定期高效表达的启动子P16。在此基础上，本研究将利用双荧光报告载体系统，对启动子P16进行生物信息学分析、结合扫描突变和随机突变分析、启动子元件功能验证等技术，确认启动子中调控稳定期特异性高效转录的顺式作用元件。利用这些元件优化启动子P16，并构建含一系列不同强度且在稳定期特异表达的启动子文库，用于表达不同的目的基因。该研究对简化生产工艺和降低生产成本具有重要应用价值，并为枯草芽孢杆菌稳定期生理调控机制研究提供理论基础。

中文关键词： 启动子体系优化；转录调控；顺式作用元件；启动子文库；原核表达系统

英文摘要： Bacillus subtilis, is the ideal host for the production of many industrial enzymes. A strong and controllable promoter is one of the key factors for efficiently expressing heterologous protein. Stationary phase specific promoters are the ideal promoters for the genes expression system , as they could express genes without inducer. Analysis and optimization of these promoters' core elements would explain the molecular mechanism of the genes expression specifically at the stationary phase and has important theoretical significance and application prospects. The analysis of the transcriptome of B.subtilis during the growth revealed that some genes were specifically expressed during stationary phase. Among the stationary phase specific promoters，we found the promoter P16 which the transcription activity is higher than the widely used constitutive promoter P43. Therefore, this study will analyze the cis-regulatory element of the promoter P16 by scanning mutagenesis, random mutagenesis, and the two fluorescent protein genes reporter system. The library of stationary phase specific promoters which cover a range of promoter activities with these elements will be constructed. These promoters can efficiently produce heterologous proteins without inducer. This work could play an important role in simplifying the production process and reducing cost in industrial production. Meanwhile, the analysis of these cis-elements provides theoretical basis for the research of the stationary phase physiological regulation in B.subtilis.

英文关键词： The promoter system optimization;transcriptional regulation;cis-regulatory element;promoter library;prokaryotic expression system