两组分系统ResD/E调控根际促生菌解淀粉芽孢杆菌SQR9根际定殖的分子机理研究

Plant growth-promoting rhizobacteria (PGPRs) play important roles in plant growth and health, application of PGPRs as microbial fertilizers is a promising approach to reduce the chemical fertilizer input and support the sustainability of agriculture. The bacterial strains of the microbial fertilizers were challenged by the native microbiome and the complex soil environmental condition, enhancing the root colonization of the bacteria is an important factor for stabilizing their functional effects. This proposal will focused on the PGPR strain Bacillus amyloliquefacien SQR9, which was isolated by our lab and applied widely in agricultural practice as the microbial fertilizer. Our previous research found that the two-component regulatiory system ResD-ResE was related to the biofilm formation and root colonization of SQR9, but the regulation pathway and molecular mechanism need further research, which will be the work of this proposal. As the “language” of the plant to interact with other organisms, root exudates are the source of signals which regulate the interactions between bacteria and plants in rhizoshpere. This proposal also includes the screening of the signal molecules in the cucumber root exudates, which have the effect on the ResD-ResE system of SQR9, and investigate their effects on the root colonization of SQR9. This research will provide the theoretical instructions for improving the root colonization and agricultural application of B. amyloliquefacien SQR9.

根际促生菌对植物生长和健康发挥重要影响，作为微生物肥料应用是实现我国化肥减施、促进农业可持续发展的有效途径。微生物肥料菌株进入土壤后面临着土著微生物的竞争及复杂的土壤环境条件影响，增强微生物肥料菌株在根际的定殖是保障其功能效果的重要因素。本申请以实验室分离并广泛应用的根际促生解淀粉芽孢杆菌SQR9为材料，在前期发现两组分系统ResD-ResE调控SQR9根际定殖成膜的基础上，深入阐释其中的调控途径及分子机理，进一步完善SQR9根际定殖成膜的分子调控网络，为深入理解以芽孢杆菌为代表的微生物肥料菌株的根际生态行为提供理论基础。根系分泌物是植物交流的“语言”，在菌-根互作中发挥重要作用。本申请还将寻找影响ResD-ResE的根系分泌物信号分子，研究根系分泌物信号分子在SQR9根际定殖中的作用。本研究将为进一步增强SQR9在根际的定殖能力、更好的发挥其农业应用效果提供应用指导。

根际促生菌对植物生长和健康发挥重要影响，作为微生物肥料应用是实现我国化肥减施、促进农业可持续发展的有效途径。微生物肥料菌株进入土壤后面临着土著微生物的竞争及复杂的土壤环境条件影响，增强微生物肥料菌株在根际的定殖是保障其功能效果的重要因素。根际的有效定殖是植物根际促生菌发挥促生和拮抗功能的前提，而生物被膜形成能力的强弱是根际定殖的关键。本项目以实验室分离并广泛应用的根际促生解淀粉芽孢杆菌SQR9为材料，通过基因敲除、定量PCR、等温滴定实验、凝胶迁移实验、DNase I footprint 实验、细菌双杂交实验等方法，研究了解淀粉芽孢杆菌SQR9中两组分调控系统ResDE识别环境缺氧信号，并调控生物被膜形成的分子机理。发现环境缺氧信号可促进SQR9菌落高级结构的形成，荧光定量PCR结果表明环境缺氧信号通过诱导胞外基质的合成促进SQR9生物被膜形成，并阐明了ResDE参与缺氧信号诱导的SQR9生物被膜形成过程。通过对不同氧分压条件下胞内NAD+/NADH比率的测定和ITC实验证明激酶ResE可通过与NAD+结合从而识别胞内NAD+/NADH比率的降低，对缺氧条件下不同突变体菌株的菌落形态观察，发现环境氧分压降低造成细胞呼吸过程受损，从而促进SQR9生物被膜的形成。两组分调控系统ResDE影响细胞色素复合物cytochrome caa3、cytochrome aa3、cytochrome bc的合成，ResD可直接调控cytochrome caa3和cytochrome aa3的合成，从而影响细胞呼吸过程。通过构建△cta、△qox和△qcr突变体菌株，发现cytochrome aa3对SQR9响应环境氧分压变化具有重要作用， cytochrome aa3参与SQR9生物被膜形成过程。对kinB基因的敲除及对该突变体在不同环境氧分压条件下菌落形态变化的观察，发现激酶KinB同样参与缺氧信号诱导的SQR9生物被膜形成。细菌双杂交实验结果表明cytochrome aa3与激酶KinB以蛋白互作的方式通过KinB-Spo0A的途径调控SQR9生物被膜的形成。本研究完整阐述了两组分调控系统ResDE识别环境氧分压降低的信号，通过细胞电子传递链调控SQR9生物被膜形成的分子机制。本项目成果将为进一步增强SQR9在根际的定殖能力、更好的发挥其农业应用效果提供应用指导.