Paenibacillus polymyxa SQR-21中杀镰孢菌素合成基因簇启动子分析与转录调控研究

Application of rhizosphere beneficial microbes in stimulation of plant growth and biocontrol of soil-borne diseases is an important strategy for suppression of soil-borne disease and decrease the input of agricultural chemicals. Paenibacillus polymyxa, an important PGPR group, could produce fusaricidins, which is the major component contributing to suppression of fungal pathogens. However, few research has focused on the transcription and regulation imformation of fus gene cluster that responsible for synthesis of fusaricidin. P. polymyxa SQR-21 was isolated by our laboratory and has been widely applied in agricultural production. In this project, the transcriptional start site of fus gene cluster, which encoding the fusaricidin synthetase, will be determined. The transcriptional activity of the promoters with different length in Escherichia coli and SQR-21 will be evaluated. The potential transcription factor that could bind to the promoter region of fus gene cluster will be screened from the intracellular protein of SQR-21 and confirmed for their binding function. Finnally, studies for improvement of the ability of SQR-21 to secret fusaricidin and suppress fungal pathogen by overexpression/knock out of these confirmed positive/nagative regulators will be carried out. This research will provide theoretical instructions and technical support for enhaning the ability of antibiotics proudction and pathogen suppression of SQR-21.

合理施用根际有益菌是克服作物土传病害、减少我国农用化学品施用的有效途径之一。多粘类芽孢杆菌（Paenibacillus polymyxa）是一类重要的根际有益菌，其产生的抗生素类次生代谢物质fusaricidin是拮抗病原真菌的主要活性成分，但有关其功能基因启动子分析与转录调控的研究还鲜有报道。本申请以实验室分离保存并已推广应用的根际有益菌P. polymyxa SQR-21为材料，确定其编码fusaricidin合成酶的fus基因簇的转录起始位点，研究其不同长度启动子在大肠杆菌和SQR-21中的转录强度。筛选SQR-21的胞内蛋白中能与该启动子区域结合的转录调控因子并验证其功能，最后通过过表达/敲除这些正/负调控因子以提高SQR-21产生fusaricidin和拮抗病原真菌的能力。本研究将为进一步增强SQR-21产生拮抗物质能力的工作提供理论依据和技术支持。

合理施用根际有益菌及其生物肥料是克服作物土传病害、实现我国化肥减施的有效途径之一。多粘类芽孢杆菌（Paenibacillus polymyxa）是一类重要的根际有益菌，其产生的次级代谢产物脂肽类抗生素fusaricidin是拮抗病原真菌的主要活性成分，但有关其功能基因启动子特征与转录调控的研究还有待深入。P. polymyxa SQR-21是本实验室分离保存的一株根际有益菌，其对多种病原真菌均表现出良好的拮抗能力，由该菌制成的生物有机肥在盆栽和大田试验中表现出良好的促生与生防效果，并已在农业生产中得到广泛应用。本项目以菌株SQR-21为研究对象，围绕其fusaricidin合成基因簇（fus基因簇）的启动子结构与转录调控特征展开研究，获得了以下主要结果：通过生物信息学分析和5'-RACE实验初步确定了菌株SQR-21中fus基因簇的转录起始位点，解析了其启动子的结构；通过绿色荧光蛋白GFP融合表达的方法研究了fus基因簇不同长度启动子的转录活性，发现启动子的-383 到 -282 bp区域可能是一个负转录调控因子结合区，而-184与-82 bp区域可能结合一个正调控因子；通过SQR-21胞内总蛋白与fus基因簇启动子Pfus片段的pull-down实验获得了大量可能与启动子片段结合的候选转录因子；通过异源表达和凝胶阻滞实验（EMSA）证实SQR-21中调控蛋白1_11（两组分调控系统中的调控蛋白，蛋白编号为595633074）可特异性与Pfus片段结合；通过定点敲除、异源表达和EMSA实验证实SQR-21中的全局性调控因子AbrB可特异性与Pfus片段结合，敲除该基因后突变体产生fusaricidin及拮抗病原真菌的能力均显著增强。上述结果对揭示菌株SQR-21中fusaricidin合成的基因调控信息，以及提高SQR-21拮抗病原真菌的能力和达到更好的田间应用效果具有一定指导意义。未来将进一步明确fus基因簇启动子的转录结构，进一步筛选可与Pfus区域结合的转录因子并明确其对fusaricidin合成的调控特征与机制，最终为进一步增强菌株SQR-21产生抗生素和拮抗病原真菌能力、更好地发挥其生物肥料在农业生产中的应用效果提供理论依据和技术支持。