荧光假单胞菌FD6主要通过产生2,4-二乙酰基间苯三酚（2,4-diacetylphloroglucinol，2,4-DAPG）等多种次生代谢产物防治植物真菌病害，且抗生素合成受到感应激酶GacS、RetS和cAMP受体蛋白同源物Vfr的调控。前期研究发现vfr对2,4-DAPG合成有负调控作用，但调控机制还不清晰。本项目拟采用生物信息学分析Vfr可能的作用靶标，通过Vfr结合域定点突变及凝胶迁移电泳（EMSA）获得影响2,4-DAPG产生的靶标蛋白，构建相应的Vfr靶标基因的内缺失突变体、互补恢复突变，检测其对抗生素2,4-DAPG合成的影响，解析Vfr间接调控抗生素2,4-DAPG合成的途径。对以上问题的研究，不仅可明确Vfr在生防菌生防功能中的作用，对假单胞菌抗生素合成调控研究是个补充，其结果还可为生防菌遗传改良提供重要理论依据，在工业微生物发酵和医学细菌病害的研究上也有指导作用。

Pseudomonas fluorescens FD6 protects various plants against plant fungi through antibiotics 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). Sensor kinase GacS, RetS and cyclic-AMP binding protein orthologue Vfr could regulate the biosynthesis of antibiotics 2,4-DAPG, PLT and PRN. Our previous study showed that Vfr had negative effect on the biosynthesis of these three antibiotics, whereas the regulatory mechanism is still unknown. In this proposal, the possible Vfr binding domain will be obtained by bioinformatics analysis, and the Vfr-target involved the biosynthesis of 2,4-DAPG will be identified by electrophoretic mobility shift assay (EMSA) and site-directed mutagenesis. The corresponding genes will be further mutated and complemented, and the effects on 2,4-DAPG production will be confirmed by HPLC. We will reveal the mechanism of Vfr as a novel regulatory element in the complex network controlling the expression of 2,4-DAPG in Pseudomonas genus. Our findings of this project will illustrate function of Vfr for biocontrol ability in biocontrol agents, and provide a supplement to the study on regulation of antibiotic biosynthesis of pseudomonads. It will also provide important reference data for future study on microbial fermentation, medical bacterial diseases and genetic modification of biological control agents.