【作者】 张静；

【导师】 何晨阳；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2008， 硕士

【摘要】 细菌鞭毛不仅在适应环境、寻找营养和躲避不良因素伤害等过程中是一种重要的运动器官,而且在趋化、附着、毒性因子产生、生物膜形成和定殖等过程中也是一种潜在的致病因子。细菌鞭毛的生物合成受到级联调控系统的严格控制。在一些重要的动物和医学病原细菌（如铜绿假单胞、霍乱弧菌和幽门螺旋杆菌等）中,存在一个以转录调控因子FleQ和σ⁵⁴因子RpoN为主控因子的4层级联调控系统,FleQ和σ⁵⁴因子协同调控了不同类型鞭毛基因的表达。水稻白叶枯病菌（Xanthomonas oryzae pv. oryzae,简称Xoo）侵染引起的白叶枯病是世界水稻生产上最严重的细菌病害之一,也是病原物—植物互作研究的一种重要模式系统。Xoo单根极生鞭毛是重要的运动器官和潜在的致病因子。然而,至今对Xoo鞭毛生物合成的调控机理尚不清楚。对Xoo基因组进行生物信息学分析,发现了由大约60个基因组成了鞭毛基因簇,其结构排列、序列特征、转录单元和转录方向与铜绿假单胞等相似,其中存在FleQ和RpoN同源序列FleQxoo和RpoNxoo。推断Xoo可能同样存在一个以FleQxoo和RpoNxoo为主控因子的级联调控系统。本研究通过基因克隆、序列分析、缺失突变、表型测定、鞭毛基因和TTSS基因表达检测,对FleQxoo和RpoNxoo的分子特征及其功能进行了鉴定分析。研究目的和意义在于阐明FleQxoo和RpoNxoo在Xoo鞭毛运动性以及相关生物学性状表达中的作用;阐明Xoo鞭毛系统调控机制将为发展新型、有效和可持续的病害控制策略和方法提供科学依据。以Xoo野生型菌株PXO99^A基因组DNA为模板,用特异性引物进行PCR扩增,成功地获得了与GenBank中KACC10331序列完全一致的fleQxoo （1485bp）和rpoNxoo （1402bp）基因片段。FleQxoo是NtrC家族激活蛋白成员之一,具有与σ⁵⁴作用的结构域和DNA结合保守结构域（HTH）,通过结合靶基因启动子区域、激活基因的转录。RpoNxoo具有与激活子互作的结构域（AID）、核心结合结构域（CBD）和DNA结合结构域（DBD）的3个保守结构域。FleQ和RpoN在植物病原黄单胞菌（水稻白叶枯病菌、柑桔溃疡病菌、番茄斑点病菌、甘蓝黑腐病菌）中同源性较高,氨基酸序列同源性可达到81.06-98.08%。采用基因标记置换方法,将构建的基因缺失突变载体pKS-fleQG和pKS-rpoNG分别导入PXO99^A,发生同源重组和标记双交换,分别从基因组中缺失了fleQxoo和rpoNxoo,获得了△fleQxoo和△rpoNxoo缺失突变体。将构建的互补载体pHM-fleQ和pHM-rpoN分别转化到相应的突变体中,获得了互补菌株△fleQxoo::C和△rpoNxoo::C。与PXO99^A相比,△fleQxoo、△rpoNxoo和△fleQxoo+△rpoNxoo鞭毛产生能力丧失,运动性减弱,基因互补可以使之恢复;但胞外酶类（纤维素酶和木聚糖酶）活性以及对烟草叶组织的致敏性无明显改变。表明FleQxoo和σ⁵⁴可能主要参与了Xoo鞭毛生物合成及其运动性的调控。利用生物信息学方法,鉴定了44个σ⁵⁴依赖型鞭毛基因,其启动子中存在σ⁵⁴识别的保守结合域GG-（N10）-GC和FleQxoo结合位点保守序列CC-（N4）-C-（N3）-T。鉴定了3个σ²⁸依赖型基因（fliCxoo、cheAxoo和cheZxoo）,其启动子中存在σ²⁸识别的保守结合域TAAA-（N15）-GCCCGTT。利用RT-Q-PCR方法,检测了△fleQxoo和△rpoNxoo中6个鞭毛基因(包括自身基因fleQxoo和rpoNxoo、σ⁵⁴依赖型鞭毛基因fliAxoo、fliDxoo和fliLxoo、σ²⁸依赖型鞭毛基因fliCxoo)的转录。与PXO99^A相比,在所有突变体中这些鞭毛基因的表达均显著降低。表明FleQxoo和RpoNxoo不仅直接正向调控了鞭毛基因fliAxoo、fliDxoo和fliLxoo的转录,通过FliAxoo (σ²⁸)间接正向调控了鞭毛素基因fliCxoo的转录,而且还负向调控了自身基因fleQxoo和rpoNxoo的转录。此外,利用RT-Q-PCR方法,检测了△fleQxoo和△rpoNxoo中3个TTSS基因（转录调控基因hrpG、hrpXo和效应子基因hpaI）的转录。与PXO99^A相比,在所有突变体中这些TTSS基因的表达均显著增加。表明FleQxoo和RpoNxoo负向调控了TTSS基因（hrpG、hrpXo和hpa I）的转录。总之,本研究阐明了FleQxoo和RpoNxoo (σ⁵⁴)作为主控因子,在Xoo鞭毛生物合成、运动性及其基因表达中起重要的调控作用,揭示了Xoo可能存在一个以FleQxoo和RpoNxoo (σ⁵⁴)为一级调控因子、FliAxoo (σ²⁸)为二级调控因子的鞭毛基因级联调控系统。更多还原

【Abstract】 Flagellum is one of the major bacterial organelles for motility by using which bacteria swim in a liquid environment and swarm over a semisolid surface. In addition to these well-established functions, it has been demonstrated in many pathogenic bacteria that flagella play multiple roles in adhesion, biofilm formation, chemotaxis, colonization and invasion in the pathogenesis. The biosynthesis of flagella is tightly and hierarchically regulated. In some of the important animal and human pathogenic bacteria including Pseudomonas aeruginosa, Vibrio spp. and Helicobater pylori, there is a four-tiered transcription regulation hierarchy mainly controlled by the transcriptional regulator FleQ and the alternative sigma factorσ⁵⁴.Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae （Xoo） is one of the most important diseases that constrain production of this staple crop in many parts of the world. The disease system becomes one of the models to study the plant-pathogen interactions. Although it has been shown that single polar flagellum of Xoo is one of the most important factors in virulence, the regulation of bacterial flagellation is still unknown. Genomic analysis of Xoo revealed that about 60 flagellar genes are clustered, and fleQ homologue （fleQxoo） and rpoN homologue （rpoNxoo） was found. There seems to be a N-tiered transcriptional regulatory circuit with FleQxoo and RpoNxoo (σ⁵⁴), both of which function as the master regulators in Xoo.Molecular identification and characterization of fleQxoo and rpoNxoo was performed through gene cloning, sequencing, deletion and expression analysis. The goal is to better understand the role of FleQxoo and RpoNxoo in bacterial behavier （such as motility and virulence/pathogenicity） and to help develop the more innovative, effective and durable strategies for the disease control.FleQxoo and rpoNxoo was cloned from the genomic DNA of the wild-type PXO99^A. FleQxoo, an NtrC family activator protein, was structurally featured in theσ⁵⁴-interacting domain and DNA-binding domain with a helix-turn-helix motif, which serves as a cognate activator ofσ⁵⁴ in transcription from severalσ⁵⁴-dependent promoters of flagellar genes. RpoNxoo had three conserved domains including the activator interacting domain （AID）, core binding domain （CBD） and DNA binding domain （DBD）. FleQ and RpoN were found highly conserved in plant-pathogenic Xanthomonas spp. with 81.06-98.08% of amino acid homolegous, respectively.△fleQxoo and△rpoNxoo, the gene deletion mutants were constructed after a double crossover recombination event between Gentamycin resistance gene （GmR） and fleQxoo or rpoNxoo through the marker exchange. Both mutants were non-flagellated and attenuated in flagellar motility on the semi-solid medium, which can be restored by complementation with fleQxoo and rpoNxoo, respectively. Moreover, no significant changes in production of extracellular cellulase and xylanase in vitro, in pathogenicity on rice and induction of hypersensitive response （HR） on non-host tobacco were observed in both mutants compared to PXO99^A.Putative sequences with consensus to GG-（N10）-GC and CC-（N4）-C-（N3）-T of RpoNxoo- and FleQxoo-binding sites were identified from the promoters of flagellar genes. Putative sequence with consensus to TAAA-（N15）-GCCCGTT ofσ²⁸-binding sites was identified from the promoters of 3 genes （fliAxoo, cheAxoo and cheWxoo）. Real-time quantitative PCR analysis of the transcription of 6 flagellar genes （fleQxoo, rpoNxoo, fliAxoo, fliCxoo, fliDxoo and fliLxoo） in△fleQxoo and△rpoNxoo indicates that both FleQxoo and RpoNxoo not only up-regulate the transcription of fliAxoo, fliDxoo and fliLxoo directly and up-regulate fliCxoo through the control of fliAxoo indirectly, but also down-regulate theirself.In addition, 3 genes involved in TTSS （hrpG, hrpXo and hpaI） are up-regulated remarkably in△fleQxoo and△rpoNxoo compared to PXO99^A, suggesting both FleQxoo and RpoNxoo repress hrpG, hrpXo and hpaI genes.In summary, both FleQxoo and RpoNxoo as the major regulators play important roles of flagellation and motility in Xoo. It is suggested that there is likely a transcription regulation hierarchy controlled by the transcriptional regulator FleQxoo and the sigma factorsσ⁵⁴ andσ²⁸ in Xoo.更多还原