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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
Downloads: 5
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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.

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