吡咯伯克霍尔德氏菌Lyc2抗细菌关键基因的鉴定与功能分析

Bacterial wilt caused by Ralstonia solanacearum is an important soil-borne disease, and there are no effective agents to control the disease. Burkholderia species is an important class of biocontrol agents and widely used for fungi diseases management. The previous study demonstrated that B. pyrrocinia Lyc2 has significant antagonism against various pathogenic bacteria and fungi, and found that the cyclic polypeptide Occidiofungin is the main antifungal substance, but the deletion of its coding gene does not affect the inhibition of Lyc2 against bacteria, which indicated that the antibacterial activity of strain Lyc2 may be a novel substance. This project targets the phytopathogenic bacteria R. solanacearum, and screen mutants that defective or lost antibacterial ability using plate assay. The rescue cloning method will be used for mutant gene locate, clone and function analysis. Through transcriptome analysis of wild-type and mutant strains to reveal the synthetic pathway of antibacterial substances and locate the regulatory genes responsible for the synthesis of antibacterial substances. The expected results will theoretically reveal the molecular mechanism by which Lyc2 inhibits pathogenic bacteria; in practice, it can provide theoretical guidance for the development of new antibacterial drugs.

烟草青枯病是一类由青枯劳尔氏菌引起的、危害严重的细菌性土传病害，目前生产上尚无有效的防治药剂。伯克霍尔德氏菌是一类重要的生防因子，可以防治多种真菌病害。前期研究结果发现，吡咯伯克霍尔德氏菌Lyc2对多种病原真菌、细菌均具有显著的拮抗作用，环状多肽Occidiofungin是其主要的抗真菌物质，但是突变该环状多肽编码基因并不影响Lyc2对病原细菌的抑制活性，推测Lyc2中抗细菌活性物质可能是一种新的抗菌物质。本项目拟以青枯劳尔氏菌为靶标，构建Lyc2突变体库，通过平板对峙生长实验，筛选抑菌能力明显下降或丧失的突变体；利用质粒拯救方法对突变基因进行定位、克隆与功能验证；通过RNA-Seq技术对野生型菌株Lyc2与突变菌株进行转录组分析，揭示抗细菌物质合成途径；挖掘抗细菌物质合成调控基因并进行功能验证。预期结果理论上将揭示Lyc2抑制病原细菌的分子机制，实践上可为研发新的抗细菌药物提供理论指导。

伯克霍尔德氏菌是一类重要的生防因子，吡咯伯克霍尔德氏菌Lyc2对多种病原真菌、细菌均具有显著的拮抗作用，环状多肽Occidiofungin是其主要的抗真菌物质，但是突变该环状多肽编码基因并不影响Lyc2对病原细菌的抑制活性，推测Lyc2中抗细菌活性物质可能是一种新的抗菌物质。本项目主要研究内容：1）利用生物信息学手段，预测菌株Lyc2全基因组中可能负责抗菌活性物质合成的基因簇14个，分为8大类，分别为Arylpolyene类、Terpene类、Phosphonate类、Hserlactone类、Nrps 类、Bacteriocin类和Nrps-Type I PKS类；2）建立了一套基于线环同源重组的可以用于菌株Lyc2高效遗传操作的系统（pBBR1-Rha-redγ-BAS-Cm），可以用于单基因或多基因的突变、敲除研究；3）采用随机突变的方式构建了菌株Lyc2的突变体库，筛选抗菌能力丧失突变体，发现谷胱甘肽合成酶基因（gshB）的突变造成菌株Lyc2失去对病原细菌的拮抗能力，对该基因的回补试验可以恢复突变菌株的抗细菌活性，另外通过外源补充谷胱甘肽可以恢复突变菌株对病原细菌的抑菌活性；4）利用pBBR1-Rha-redγ-BAS-Cm系统对Lyc2中的gshB及关联基因进行了单基因和多基因敲除，进一步验证了gshB基因的功能；5）转录组分析结果表明，gshB基因的突变会显著影响运动性相关的基因的表达，包括鞭毛和趋化性基因都出现显著的下调，推测gshB基因的突变会影响菌株的运动性，而运动能力的受限影响了Lyc2对病原细菌的抑制能力。项目研究成果对于吡咯伯克霍尔德氏菌抗菌机制的解析和深度挖掘菌株次级代谢产物用于开发新型抗细菌病害的生物农药具有重要意义。