【作者】 陈慧；

【导师】 周莲；

【作者基本信息】 上海交通大学 ， 生物学， 2019， 硕士

【摘要】 野油菜黄单胞菌(Xanthomonas campestris pv.campestris,Xcc)是十字花科植物黑腐病的致病菌。Xcc致病性受到DSF信号依赖的群体感应系统和Rpf B介导的群体感应退出机制调控。本研究分别检测了18种氨基酸对DSF-家族群体感应信号分子合成的影响,并初步探究其中有效果的三种氨基酸的最佳作用条件、生物学功能和作用机理。研究中采用的主要实验方法包括添加不同浓度的氨基酸到ΔrpfC菌株XYS培养体系中,接种后不同时间点取样提取DSF信号分子,利用高效液相色谱法(HPLC)分析DSF和BDSF浓度;以及利用gus A报告菌株和Western blotting检测甲硫氨酸对rpf B和rpf F表达水平的影响。研究结果表明1)18种氨基酸中,甲硫氨酸,色氨酸和胱氨酸能有效降低ΔrpfC菌株培养体系中DSF和BDSF水平,抑制效果与氨基酸浓度密切相关;2)三种氨基酸对DSF信号分子的抑制作用存在叠加效应;3)甲硫氨酸、色氨酸或胱氨酸不影响ΔrpfCΔrpf B双突变体菌株中DSF和BDSF水平;4)甲硫氨酸对降解酶基因rpf B的转录水平和翻译水平没有影响,但显著抑制合成酶基因rpf F的转录和翻译;5)其他DSF信号途径的关键组分(Clp和Rpf G)不参与甲硫氨酸诱导ΔrpfC菌株XYS培养体系DSF和BDSF水平降低过程;6)在NYG、LB和NA等营养丰富的培养基中,甲硫氨酸不影响ΔrpfC菌株培养体系中DSF和BDSF水平;7)S-腺苷甲硫氨酸也能有效降低ΔrpfC菌株培养体系中DSF和BDSF水平。本研究鉴定了三种有效抑制DSF信号合成的氨基酸并初步探究了其作用机理;不仅拓宽了对病原菌与植物互作机制的认识;也为以病原细菌的群体感应为靶标设计新型植物病害防治方法提供了思路。更多还原

【Abstract】 Xanthomonas campestris pv.campestris(Xcc)is a casual agent of black rot in cruciferous plants.The diffusible signal factor(DSF)-dependent quorum sensing(QS)system and Rpf B-dependent QS exit mechanism are closely associated with Xcc pathogenicity.we investigated the effects of 18 amino acids on the biosynthesis of DSF-family QS signals,and the optimum conditions,biological functions and mechanisms of three effective amino acids were preliminarily explored.And we used the gus A reported strain and western blotting to investigated the effect of methionine on transcription and translation of Rpf B or Rpf F.The results show that:1)Among the 18 amino acids tested,methionine,tryptophan and cystine were found to significantly decrease DSF and BDSF levels in theΔrpfC culture.The inhibitory effect was closely associated with amino acid concentration;2)Additive inhibitory effects between three amino acids were observed;3)Addition of methionine,tryptophan or cystine had no significant effect on the levels of DSF and BDSF of theΔrpfCΔrpf B culture;4)Methionine had no effect on the transcription and translation of the degrading enzyme gene rpf B,but significantly inhibited the transcription and translation of the synthase gene rpf F;5)Other key components of DSF signaling pathway(Clp and Rpf G)were not involved in the process of methionine-induced decrease of DSF and BDSF levels in XYS culture system ofΔrpfC strain;6)In nutrient-rich media such as NYG,LB and NA,methionine did not affect the levels of DSF and BDSF in the culture system ofΔrpfC strain;7)S-adenosylmethionine can also effectively reduce the levels of DSF and BDSF in the culture system ofΔrpfC strain.Our study identified the effective amino acids against DSF of Xcc and preliminarily explored their mechanisms.It not only broadens the understanding of the interaction mechanism between pathogens and plants,but also provides ideas for designing new plant disease control methods based on quorum sensing of pathogenic bacteria.更多还原