The Function Identification Of Two Genes Which Code Putative Protein In Xcc 8004

The Xanthomonas campestris pv.campestris（Xcc）,also known as the cabbage black rot bacteria,is gram-negative bacteria,which can cause worldwide black rot disease in cruciferous plants.It mainly infects the cabbage,rape,Chinese cabbage,shepherd’s purse,radish and other crops,thus brings great damage to agricultural economy.Form the previous research,we have screened a group of genes which are possibly related to Xcc 8004 pathogenicity,extracellular polysaccharide（EPS）synthesis.In order to further identify their function,we used the suicide plasmid pK18mobSacB and pK18mob to construct deletion mutant and non-polar integrated mutant of XC₁348 and XC₃744 by homologous double-crossover and homologous single-crossover respectively.Then we analyzed and compared the phenotype of these mutants.According to Xcc 8004 genome annotation,XC₁348 codes a putative protein,phenotypes of the deletion mutant and non-polar integrated mutant of XC₁348 showed that the mutation on XC₁348 gene had no effect on the production of EPS,the activity of extracellular enzyme,bacterial motility and basic growth.However,the mutants can cause hypersensitive response to the non host plant.Compared to wild-type strain,the deletion mutant had similar virulence to the wild-type strain while the pathogenicity of integrated mutant decreases significantly.The virulence of two mutants showed great difference in host radish,therefore we construct the complementary strain CNK1348 of integrated mutant and tested its pathogenicity.The pathogenicity of CNK1348 cannot be restored to wild-type level,which indicates that the XC 1348 gene had no effect on pathogenicity.Using the quantitative RT-PCR method to analyze the mutual relationship of putative gene clusters consisted of XC₁348,XC₁347,XC₁346 and XC 1345 at the transcription expression level,the result showed that XC₁348 negatively regulates three downstream genes.Gel retardation experiment showed that the protein coded by XC₁348 is a transcriptional factor.According to Xcc 8004 genome annotation,XC₃744 codes a putative protein,the pathogenicity of deletion mutant decreased significantly,but the complementary strain CDM3744 can restore its pathogenicity to 90%of the wild-type level,which indicated that XC₃744 is related with pathogenicity.The ability of mutant resistant to protein denaturant decreased,but the production of EPS,the activity of extracellular and the motility are not affected.The result of 5’RACE experiment showed the transcription initiation site is at 55 bp upstream of XC₃744.