地衣芽孢杆菌HG03诱导番茄系统抗病关键外泌蛋白的鉴定及生物学功能研究

Previous studies found that the crude extracts of secreted proteins from Bacillus licheniformis HG03 could induce systemic disease resistance in tomato. This project is proposed on the basis of previous studies. By the methods of ammonium sulfate precipitation, ion exchange chromatography and mass spectrometry, HG03 secreted proteins will be separated, purified and identified to obtain the key proteins with the function of inducing systemic resistance in tomato. On the basis of the whole genome sequence information of HG03, the protein-coding genes will be cloned, and following with prokaryotic expression and purification. The biological function of the purified proteins will be tested in indoor and field experiments, and the phenotype and the related gene expression in tomato will also be verified. Through yeast two-hybrid system, the proteins in tomato roots that can interact with the secreted proteins from HG03 will be screened, and the selected proteins are verified with pull-down assay. So the molecular mechanisms of the HG03 secreted proteins inducing disease resistance in tomato will be analyzed, and so as B. licheniformis HG03. This research will lay the foundation for the application of the biocontrol agent B. licheniformis HG03. The results will provide theoretical basis and support for the application of B. licheniformis HG03.

前期研究发现，生防菌地衣芽孢杆菌（Bacillus licheniformis）HG03的外泌蛋白可以诱导番茄系统抗病性产生，对多种番茄病害具有较好防效，但是生防作用机理仍不清楚。本项目拟在前期研究基础上，分别利用硫酸铵沉淀、离子交换层析和质谱分析等方法对HG03的外泌蛋白进行分离、纯化和鉴定，得到具有诱导番茄系统抗病性功能的关键蛋白；结合已获得的HG03全基因组序列信息，克隆关键外泌蛋白的编码基因，进而对其进行原核表达和纯化。通过温室实验验证表达蛋白的生物学功能，检测其诱导番茄系统抗病性表型。利用酵母双杂交系统筛选与HG03关键外泌蛋白存在互作关系的番茄根系相关因子，并通过pull-down assay加以验证，从而解析HG03关键外泌蛋白诱导番茄抗病性的分子机制。该研究结果将为生防地衣芽孢杆菌（B.licheniformis）HG03更好的开发应用提供理论基础和技术支持。

生防菌地衣芽孢杆菌（Bacillus licheniformis）HG03较好的生防潜力，其外泌蛋白可以诱导番茄系统抗病性产生，对多种番茄病害具有较好防效，但是生防作用机理仍不清楚。本项目在前期研究基础上，对生防菌地衣芽孢杆菌（B.licheniformis）HG03的外泌蛋白进行分离、纯化和鉴定，得到了具有诱导番茄系统抗病性功能的关键蛋白；结合HG03全基因组序列分析，克隆关键外泌蛋白的编码基因，进而对其进行原核表达和纯化；利用酵母双杂交系统筛选与HG03关键外泌蛋白存在互作关系的番茄根系相关因子，并通过pull-down assay加以验证，对HG03关键外泌蛋白诱导番茄抗病性的分子机制进行了解析。该研究结果将为生防地衣芽孢杆菌（B.licheniformis）HG03更好的开发应用提供理论基础和技术支持。