Construction,screening Of Pseudomonas Syringae Pv. Tabaci EZ::Tn5 Mutants And Mutated-gene Cloning

Construction of mutant library of plant pathogenic bacteria is an important and indispensable component of functional genomics research, and lays the foundation of revealing important genes.Tobacco wildfire disease, caused by Pseudomonas syringae pv. tabaci, is one of the most important bacterial disease. The pathogenic bacteria is a typic pathovar of Pseudomonas sryingae-group in which more than 50 pathovars identified, less molecular and genomic researches were carried out on it than P. syingae pv. tomato , P. syingae pv. syringae and P. syingae pv. phaseolicola. In order to explore pathogenicity related genes of P.syingae pv.tabaci, the strategy of transposon-mediated mutagensis was used in this study. The EZ::Tn5 and kanr genes carried in Tnp TransposomeTM were introduced into tobacco wildfire pathogen genomics by electrical Transformation, and then the EZ::Tn5 random insertion mutant library was constructed. Mutants with mutated pathogenicity, motility and extracellular protease production ability were screened. EZ::Tn5 insertion site flanking sequences were amplified by TAIL-PCR. The goal of this research is to get ideal materials for analyze bacteria-host molecular interactions. The major results are as follows:1. By the electroporation, the EZ::Tn5 transposition system were successfully inserted into P. syringae pv. tabaci strain WT4( tabtoxin producing strain) and strain SMX006 (tabtoxin defective strain) respectively. And their EZ::Tn5 insertion mutant libraries were constructed. Under the condition of 2400 voltage, the electroporation efficiency of strain WT4 was 4.2×103cfu.μg-1 DNA , and that of SMX006 was 0.8×103 cfu.μg-1 DNA. While the voltage was 1800V, the electroporation efficiency of WT4 decreased down to 1.64×103 cfu.μg-1 DNA. By specific PCR identification, Kan resistance screening and successive transfer culturing, it was indicated that the resultant mutants had stabile EZ::Tn5 insertion into the pathogen genomic DNA.2. Assays of pathogenicity, motility and extracellular protease activity on SMX006 EZ::Tn5 mutants were carried out. And 2 mutants (H028,H029) with significantly reduced pathogenicity, 1 mutant (H028) with weaker motility, 1 motility-defective mutant (H029), 2mutant (H046,H047) with significantly decreased extracellular protease activity, and 1 extracellular protease deficient mutant (H045) were screened out.3. In order to determine the insertion copy of EZ::Tn5 mutant, the digoxin-labeled DNA probes were synthesized from the EZ::Tn5-specific PCR primers. 4 SMX006-derived mutants (H028,H029,H05,H046) with changed-phenotype and 4 SMX006-derived mutants (H025,H031,H048,H071) lack of obvious phenotypic changes were selected, their genomic DNA was digested with HindⅢrestriction enzyme, respectively. The results southern hybridization showed that all the tested EZ::Tn5 mutant DNA has only one EZ::Tn5 transposon insertion.4. By TAIL-PCR method, flanking sequence of EZ::Tn5 insertion site of 8 tested mutants were amplified and sequenced. Sequence blasting showed that 7 of 8 mutants had EZ::Tn5 insertion into 23SrRNA or 16SrRNA gene. Only one mutant was non-rRNA insertion.