生防假单胞菌新型抗菌肽类物质鉴定及RpoS对其生物合成调控的分子机制

生防假单胞菌FD6主要通过产生抗生素2,4-二乙酰基间苯三酚和藤黄绿脓菌素防治番茄灰霉病。前期研究发现菌株FD6在土豆浸汁培养基中产生的抗菌物质对番茄灰霉病菌具有较强抑菌活性，且其分子量不同于已报道的脂肽化合物，说明该物质可能为新型脂肽类抗菌物质。为明确该抗菌物质种类，本项目拟将硅胶柱粗分离得到各极性组分，以核磁共振氢谱和薄层层析示踪，组合使用多种层析及高效液相色谱等方法对组分进行分离纯化，进一步采用高分辨质谱和核磁共振谱等现代波谱技术及文献比对的方法鉴定未知抗菌物质的化学结构，通过基因缺失突变及功能互补，以期明确主要抗菌物质及其相关合成基因簇；采用ChIP-Seq、EMSA及qPCR等方法，阐明sigma因子RpoS调控抗菌脂肽合成的机制。研究结果将为今后生物农药开发提供先导化合物，并有望在假单胞菌中揭示RpoS行使调控功能的一种新机制。

The production of antibiotics 2,4-diacetylphloroglucinol and pyoluteorin are the key biocontrol factors involved in tomato gray mold disease controlled by Pseudomonas protegens FD6. Previous studies showed that antifungal extracts from P. progegens FD6 cultured on PA media displayed stronger antagonistic activity against Botrytis cinerea. Morover, the molecular weight of the antifungal compound is different from those antimicrobial peptides produced by pseudomonad, suggesting that P. progegens FD6 may produce new antifungal metabolites under nutrient deficient conditions. To identify the antifungal metabolites, the ethyl acetate extract of P. progegens FD6 are chromatographed on silica gel column and several different polar mixtures will be obtained. Guided by 1H-NMR spectrum and thin-layer chromatography, further separation and purification are performed using several methods such as silica gel column chromatography, Sephadex LH-20 gel column chromatography and high performance liquid chromatography (HPLC). Finally, structures of these compounds will be determined by the modem spectrum technology such as HR-ESI-MS, 1D and 2D NMR comparing with references. The main antimicrobial substances and the gene cluster responsible for the biosynthesis of the cyclic lipopeptides will be identified through gene deletion mutation and functional complementation. This project will reveal the regulatory pattern of sigma factor RpoS on the cyclic lipopeptide biosynthesis using ChIP-Seq、EMSA and qPCR. The outcomes of the project are expected to provide lead compounds for the development of new drugs and reveal a new RpoS-mediated regulatory mechanism in Pseudomonas, or even in bacterial level.