【作者】 周洁；

【导师】 唐纪良； 唐东阶；

【作者基本信息】 广西大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 Omega(ω))亚基是细菌RNA聚合酶(RNA polymerase)最小的一个亚基,尽管它在细菌中广泛存在并且序列非常保守,但由于在许多细菌中编码ω亚基的基因突变后没有明显的表型变化,因此人们通常认为它是不重要的。到目前为止,在大多数细菌(包括动植物病原细菌)中ω亚基的功能还是未知的。十字花科黑腐病菌(Xanthomonas campestris pv.campestris,简称Xcc),是引起十字花科植物黑腐病的病原菌;它是研究病原细菌与植物之间复杂相互作用的模式菌之一。基因组注释结果表明,十字花科黑腐病菌8004菌株(Xcc 8004)基因组中存在一个编码ω亚基的基因rpoZ(ORF编号为XC0913基因)。为研究ω基的功能,本工作采用自杀质粒pK18mob突变方法,构建了该基因的非极性突变体NK0913。表型检测结果表明:(1)NK0913在丰富和基本培养基上的生长均比野生型菌株明显减慢;(2)NK0913在非寄主植物辣椒ECW-10R上不能产生HR反应而野生型菌株8004在该植物上能产生正常的HR反应,在寄主满身红萝卜上的致病力明显低于野生型菌株8004;(3)NK0913的胞外多糖产量、胞外酶(纤维素酶、蛋白质酶和淀粉酶)的活性、细胞运动能力比野生型菌株显著降低;(4)NK0913在LB培养基中表现为聚集生长而野生型菌株在该培养基中表现为分散生长。将带有完整rpoZ基因的质粒pLALR3导入ω亚基的突变体NK0913后能将该突变体的表型恢复到野生型水平。这些结果表明,ω亚基在Xcc的生长、致病、EPS合成、胞外酶产生、细胞运动、生物膜形成等过程中有重要作用。为了进一步了解ω亚基在上述生理过程中的具体作用,我们用β-葡糖醛酸酶(GUS)活性测定和RT-PCR的方法检测ω亚基与hrp基因的调控关系。GUS报告质粒结果显示,rpoZ基因的突变导致hrp基因簇中hrpB、C、F转录单元以及hrp调控基因hrpG和hrpX的表达明显下降,而RT-PCR结果显示,hrpB5、hpaP、hrcQ,hrpD6、hpaA、hpaB、hpal、hpa2和hrpW基因表达明显下降,但hrpF表达增高。根据这些结果,我们推测ω亚基可能是通过调控hrp基因的表达来影Xcc的致病性和HR反应的。我们还用RT-PCR的方法检测了ω亚基与鞭毛合成相关基因的调控关系。结果显示,rpoZ基因的突变后,鞭毛合成相关基因motA、fliD、fliE、fliL、基因的表达明显下降,表明ω亚基可能是通过调控这些基因的表达来影响Xcc的运动能力。更多还原

【Abstract】 Omega(ω),the smallest subunit of bacterial RNA polymerase(RNAP),is encoded by rpoZ,and is wildely distributed and highly conserved in various bacteria species.Since few phenotypic alterations were obsurved in the rpoZ mutant,it is belived thatωis not important in bacteria.For this reason,up to date,the exact role ofωin bacteria including animal and plant bacterial pathogens is largely unknown.Xanthomonas campestris pv.campestris(here after Xcc)is the causal agent of the black rot disease of cruciferous plants,and is the model bacteria for studying the interaction between pathogen and its plant host.Genome sequencing and annotation showed that,in the genome of Xcc strain 8004 (Xcc8004),the gene rpoZ(ORF number XC0913)encodes theωsubunit of the Xcc RNAP.To investigte the function of the co subunit of Xcc RNAP,in this study,the nonpolar mutant of rpoZ gene,named NK0913,was constructed by homologous suicide plasmid pK18mob integration and the phenotypes of the mutant were analyed.The results showed that,(ⅰ)NK0913 shows a reduced growth rate in the rich medium NYG and the minimal medium MMX compare to the wild type strain 8004;(ⅱ)NK0913 can not induce a hypersensitive reaction(HR)in the nonhost plant pepper ECW-10R,and a significant reduced virulence in the host plant Chinese radish(Raphanus sativus L.var.radiculus Pers.);(ⅲ)NK0913 displyed a reduction in exopolysaccharides(EPS) production,extracellular enzyme(cellulose,protease and amylase)activity and cell motility;(ⅳ)although the wild type train does not form aggregates in LB medium,NK0913 grows in aggregated fashion in the medium.Introduction of the plasmid pLALR3 carrying rpoZ gene into NK0913 can restore all of the phenotypes of the mutant.,These results reveal that theωsubunit plays an important role in cell growth,pathogenesis,EPS production,extracellular enzyme activity and cell motility of Xcc.To investigate the exact role ofωsubunit,an RT-PCR and the GUS-promoter transcriptional fusion reporter analysis were performed to determine the regulatory relation between rpoZ gene and hrp genes.The result of GUS-promoter analysis showed that the expression of the hrp operons hrpB, hrpC and hrpF,and the two key hrp regulatory genes hrpG and hrpX is singnificantly reduced in the rpoZ mutant NK0913.The RT-PCR results showed that the expression of hrpB5、hpaP、hrcQ,hrpD6、hpaA、hpaB、hpa1、hpa2 and hrpW is reduced but the hrpF is inncreased in NK0913.These results suggested thatωsubunit is involved in virulence and HR through regulating the expression of hrp genes. We further used RT-PCR analysis to investigate the regulatory relation between rpoZ and the genes involved in flagellum synthesis.The results showed that the expression of motA,fliD,fliE and fliL is reduced in the rpoZ mutant NK0913,indicating thatωsubunit is involved in cell motility through regulating the expression of these flagellum synthesis-related genes.更多还原