The Phenotype Screening And Mutation Site Localization Of The Tn5Mutants Of Xanthomonas Oryzae PV. Oryzicola

Xanthomonas oryzae pathovar oryzicola (Xoc), the causal agent of bacterial leaf streak (BLS) of rice (Oryza sativa), constrains production of this staple crop in parts of Guangxi. By the success of the sequencing of rice and Xoc the Philippines BLS256, interwork of Xoc-rice has become one of the modes to study interactions of pathogen-plant. The sequencing of Xoc GX01strain has almost been completed, and dynamic pathogenic bacteria biological functions are waiting for being excavated from the abundant information of statical sequences. As is known to all, the mutant library is the foundation of functional genomics research. The traits of mobility of transponson and transposition in vitro are widely used in construction of insertion mutant library. It has become a powerful tool of studying Xanthomonas.This study used Guangxi strain Xoc GX01as the original strain to construct the mutant library. By using EZ-Tn5TM<R6Kyori/KAN-2> Transposon, The Tn5transponson insertion mutant library has been constructed. Through the method of electrotransformation,2965monoclonal transformants have been got and conserved. Testing them in phenotypes of EPS, extracellaluar proteases, motility and biofilm, the study obtained the mutants that were interested us. At present,2200mutants were screened, to which the following mutants were drawn attention.26EPS secretion enhanced mutants and31EPS secretion reduced mutants,77extracellular protease activity heightened mutants,16extracellular protease activity weakened mutants,61Motility increased mutants,507motility attenuated mutants, and67mutants in biofilm phenomenon.To further assure the insertion of Tn5, we randomly chose17mutants from the mutant library, extracted DNA respectively, did polymerase chain reaction (PCR) with validated primers. If a product of586bp were got, the mutant that was inserted with Tn5was verified. What’s more, by the method of plasmid rescue, flanking sequence of mutants were also obtained. Aligning the flanking sequences and genome of GX01through software of local BLAST, it found that the similarities were more than97%. The result showed that plasmid rescuing is an effective and credible method to get the flanking sequence of mutants.The study chose BH90and BA87mutants, whose phenotypic changes were relatively obvious, to assay their virulence. The result displayed their virulence decreased to some extent separately. The locating information of their positions is XOC2200, XOC2216. At the same time, deletion mutants and its complementary strains of XOC2200, XOC2216were constructed. The product of XOC2200is transcriptional regulator, flagellar regulatory protein A. The product of XOC2216is the flagellar biosynthesis hook protein. The extracellular polysaccharide and motility of BH90and XOC2200were increased obviously. Complementary strains phenotypes could almost recovered to that of wild type. This result indicated that XOC2200negatively regulated extracellular polysaccharide synthesis and motility. Extracellular protease activity of BA87and XOC2216were enhanced, but the motility was weakened. It is showed that XOC2216influenced the extracellular protease activity and motility.