Dissertation
Dissertation > Agricultural Sciences > Plant Protection > Pest and Disease Control > Plant diseases and their prevention > Transgression ( pass ) an infectious agent harmful

Select of Biofilm Formation Mutants and Cloning and Primary Function Analysis of tatC Gene from Pseudomonas Fluorescens Strain 7-14

Author KangYueJing
Tutor LiuFengQuan
School Nanjing Agricultural College
Course Plant Pathology
Keywords Transposon mutagenesis Biofilm formation Gene cloning tatC gene Homologous recombination Function analysis
CLC S432.4
Type Master's thesis
Year 2010
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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.

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