欧美杨细菌性溃疡病菌双组分基因缺失突变及其生物学表型分析

Deletion mutations and its phenotypic analysis of two-component genes in Lonsdalea populi

【目的】欧美杨细菌性溃疡病是革兰氏阴性细菌Lonsdalea populi引起的杨树枝干病害,其危害严重,已造成欧美杨人工林的重要经济损失。双组分系统是细菌致病过程的关键调控途径之一。目前,欧美杨细菌性溃疡病菌的双组分系统如何调控致病过程仍缺乏系统研究。因此,本研究开展欧美杨细菌性溃疡病菌的双组分编码基因的缺失突变及突变体表型分析,为深入解析其致病机制提供遗传材料。【方法】本研究以欧美杨溃疡病菌菌株N-5-1为研究对象,利用双亲结合方法获得了28个双组分系统基因的缺失突变体,并通过表型测定方法分析了这些基因突变体的致病性、生长、游动性、生物膜形成和抗逆性等表型特征,研究不同双组分系统编码基因对该病菌致病过程的调控。【结果】构建了36个欧美杨溃疡病菌的双组分编码基因的敲除重组载体,获得了28个基因的缺失突变体。致病性测定表明18个双组分基因的敲除降低了病原菌的毒性,其中8个突变体毒性丧失。此外,还获得了调控游动性和生物膜形成能力的突变体以及在逆境胁迫反应(金属离子、盐离子、抗生素等胁迫)有缺陷的突变体。【结论】本研究获得了5个显著影响欧美杨细菌性溃疡病菌毒性及其他生物表型的双组分基因,为后续双组分信号调控致病机制研究提供了遗传材料。

[Objective]The bacterial canker of Populus euramericana is caused by the Gram-negative bacterium Lonsdalea populi.The rapid spread of the disease has seriously threatened the growth and development of P.euramericana and has caused great economic losses to the plantation.Two-component system(TCS)is the important signal transduction mechanism of Lonsdalea populi.Now,how the two-component system of poplar bacterial canker regulates the pathogenic process is still lack of systematic research.Therefore,the large-scale deletion mutation and mutant phenotype analysis of TCS in this study will provide genetic materials for further study on the pathogenic mechanism of poplar bacterial canker.[Method]In this study,28 two-component gene deletion mutants of poplar canker pathogen strain L.populi N-5-1 were constructed by parental association,the differences in pathogenicity,growth,motility,biofilm formation and resistance of these mutants were analyzed by phenotypic analysis,and the regulation of two-component system coding genes on the pathogenicity of these mutants was studied.[Result]In this study,36 recombinant vectors of two-component coding genes were constructed and 28 deletion mutants were obtained.Phenotypic analysis showed that 18 genes encoding TCS were involved in virulence of L.populi N-5-1.Among them,the pathogenicity of 8 mutants had obviously disappeared.In addition,the deletion mutants regulating motility and biofilm-forming ability and those deficient in stress response(metal ions,salt ions,antibiotic stress,etc.)were also screened.[Conclusion]In this study,five two-component genes significantly affecting the pathogenicity of L.populi were obtained,providing genetic material for future studies on the pathogenic mechanism of L.populi.