Cloning And Fuctional Study Of Key Genes Related To Histidine Metabolism And A Gene Related To Salt-tolerance In Xanthomonas Oryzae Pv. Oryzicola

Xanthomonas oryzae belongs to Xanthomonas,including Xanthomonas oryzae pv.oryzae?Xoo?and Xanthomonas oeyzae pv.oryzicola?Xoc?,which induce bacterial leaf blight?BLB?and bacterial leaf streak?BLS?on rice leaves.In this study,sequence analysis cloning of these genes were used in this study,and constructed these deletion mutants using the principle of homologous recombination.Then infer the corresponding function of genes by the phenotype difference between wild type strain Rs105 and mutant strains.?I?Analysis of metabolic pathway and gene define showed that the genes related to histidine biosynthesis and histidine degradation,which we called his gene cluster and hut gene cluster,were existed in Xoc.Homology analysis of these genes showed that all the two gene clusters were highly conserved in Xanthomonas,and then we got all the mutant strains by gene deleted.The results of pathogenicity on host rice showed that these mutations related to hut gene cluster had no effect on the pathogenicity on host-rice.However the pathogenicity of all the mutations belongs to his gene cluster were lower than wild type,and AhisB showed the most obvious among these mutations.All the mutant strains of his gene cluster showed a nomal ability to induce hypersensitive responses in non-host tobacco.Based on the analysis of histidine deficiency in the basal medium,we further demonstrated that the importance of his gene cluster in histidine biosynthesis of Xoc.And studies showed that hisB gene which encodes a bifunctional enzyme implicated in histidine biosynthesis,is required for virulence and yellow pigment production in Xoc.The disruption of hisB reduces the transcriptional expression of five known virulence-associated genes?wxocB,purF,pgK,tal-C10c-like,and trpA?and four known shikimate pathway-related genes?xanB2,aroE,aroA,and aroK?in XOM3 medium supplemented with 250 ?M L-histidine.Therefore,the novel roles of histidine biosynthetic pathway-related gene of hisB in virulence and yellow pigment production in Xoc are revealed for the first time.?II?Organisms have a certain tolerance to environmental factors,and their survival and reproduction will be affected if the surrounding environmental factors exceed the tolerance level of them.In nature,pathogenic bacteria also often encounter different stress coming from the environmental,including physics,biology,chemistry and other related factors will have a different effects on the metabolisms of bacteria and their activities of related enzymes,which promote the bacteria to evolve physiological and genetic mechanisms to improve its environment adaptability and make sure they can infect the host and reproduction successfully.So exploring these mechanisms involved in above effects can provide a theoretical basis for the further exploration of the regularity of microbial variation,as well as the elucidation of the mechanisms of resistance to temperature and other physical and chemical factors.In Xanthomonas oryzae pv.oryzae PX099A strain,we found a gene?PXO 03841?,which significantly reduced the activity of the strain in NaCl environment if this gene was delated.Homologous alignment showed that the amino acid sequence of the gene was highly conserved in Xanthomonas,and we found out the homologous gene?ACU12₀0945?in Xanthomonas oryzae pv.oryzicola Rs105 strain.We constructed the deleted-mutation of this gene in Rs105,and determined its genetic phenotype in order to clear the gene function.Results of the experiment showed that the ability of ACU12₀0945 mutation to tolerate NaCl was also reduced.In this experimentation,we take ACU12₀0945 as the basis gene for our further study.qRT-PCR results suggested that the expression of ACU12₀0945 gene increased with the increase of NaCl concentration in Rs105 wild-type strain,and proved that the gene was positively correlated with the salt tolerance of Rs105.The ability of the deleted mutation to tolerate KC1,was also weaker than wild-type strain.However,there was no significant difference in the growth ability between mutation and wild-type strain as the presence of CaCl2 and ZnCl2.The results showed that the ACU12₀0945 gene may be related to the sodium&potassium ion transport of intracellular,which regulate the balance of cell osmotic pressure.The study also found that the deletion of ACU12₀0945 gene in Rs105 strain reduced the swarming ability.