【作者】 康越景；

【导师】 刘凤权；

【作者基本信息】 南京农业大学 ， 植物病理学， 2010， 硕士

【摘要】 利用mariner转座子对荧光假单胞菌7-14(Pseudomonas fluorescens 7-14, Pf7-14)进行转座诱变,成功构建了Pf7-14的突变体文库。利用96孔板筛选法,从8500多株突变体中筛选得到一株生物膜增强突变株4B5和一株生物膜减弱突变株25C11。通过任意PCR (arbitrary polymerase amplification reacrion, arbitrary PCR)和亚克隆技术鉴定出,4B5的转座子插入的位点与P. fluorescens Pf0-1编码chemotaxis sensory transducer的基因,与P. fluorescens Pf-5编码methyl-accepting chemotaxis transducer的基因具有很高的同源性,25C11的转座子插入的位点与P. fluorescens Pf-5和Pf0-1的编码lipoyltransferase的基因lipB高度同源。根据Pf0-1和Pf-5相关基因的序列设计简并引物,从Pf7-14中扩增出了对应的基因。油菜离体叶片的菌体分布试验表明,和Pf7-14野生型相比,4B5在油菜叶片上粘附的细胞数量较多,25C11粘附的细胞数量较少。根据荧光假单胞菌Pf-5和SBW25的tatC基因序列设计简并引物,采用PCR方法从Pf7-14中克隆了tatC基因,命名为tatCPf7-14。通过序列分析,我们比较了tatCPf7-14与同种、同属和其它相关细菌中tatC基因的核酸序列的同源性,以及TatCPf7-14与这些细菌中TatC蛋白的氨基酸序列的同源性。通过同源重组单交换的方法,构建了Pf7-14 tatC基因的插入突变株。研究发现,(1)tatC突变株的蛋白酶、嗜铁素的产量较野生型有所降低；(2)tatC突变体的游动能力和生物膜形成能力均弱于野生型。互补菌株均恢复了上述性状。tatC基因的突变未改变Pf7-14在LB培养基中的生长速率。平板拮抗试验显示,突变体对主要病原真菌仍具有很好的拮抗能力。更多还原

【Abstract】 The mutant library of Pseudomonas fluorescens 7-14 (Pf7-14) was successfully constructed by mating mariner transposon into strain Pf7-14. We obtained 2 biofilm mutants from 8500 mutants. Compared to the wild-type,4B5 enhanced the ability of forming biofilm and 25C11 showed biofilm-defective phenotype. By Arbitrary PCR and sub-cloning, we ensured the locations of the genes inserted by transposon. The gene in 4B5 is homologous to the gene encodes chemotaxis sensory transducer in P. fluorescens Pf0-1 and the gene encodes methyl-accepting chemotaxis transducer in P. fluorescens Pf-5. The gene in 25C11 is homologous to lipB encoding lipoyltransferase in Pf0-1 and Pf-5. We cloned these two genes from Pf7-14 by PCR with degenerated primers. We observed bacterial cells distribution on isolated rape leaf assay. The adhersion ability of Pf7-14 is stronger than 25C11, and weaker than 4B5.According to the sequences of tatC gene from P. fluorescens Pf-5 and SBW25, a pair of degenerate primers was designed and used to amplify the tatC gene from P. fluorescens strain 7-14 (Pf7-14) (named as tatCPf7-14)-By sequence analysis, we compared the homology of tatCPf7-14 and TatPf7-14 to that in other kinds of bacterias. Pf7-14 tatC insertional mutantΔtatC has been constructed by single homologous recombination. Phenotypes of tatC gene mutant has been studied:(1)ΔtatC decreased the production of protease and siderophore; (2)ΔtatC was defective in swimming motility and biofilm formation; (3) The mutant affected in the tatC gene did not change the gowth rate when cultured in LB; (4)ΔtatC had the same phenotype in the antifungal activity to Rhzoctonia solani and Sclerotinia sclerotiorum. Complementation of tatC mutant restored all altered phenotypes.更多还原