The Construction Of MCP Free Strains And Characterization Of MCP Genes In Xanthomonas Oryzae Pv.oryzae PXO99~A

Xanthomonas,most of which are plant pathogenic bacteria in natural environment,can infect many kinds of plants which are the source of human food and cause symptoms such as leaf blight or leaf spot.The Xanthomonas oryzae pv.oryzae（Xoo）strain studied in this experiment is a pathovar of Xanthomonas.This strain is short rod-shaped,has a polar monoflagellum.It’s gram-negative and has no spore or capsule.It is surrounded by extracellular polysaccharides.Xoo infects rice through the leaf wound or the hydathode,and then there will be grey disease spot growing along its vascular bundle.This strain can cause a severe reduction of rice every year in China.As a result,it is necessary to study Xoo.The mechanism of pathogenicity of Xoo has been studied a lot in recent years.However,the reason why this strain can colonize and infect rice still keeps unclear,and methyl-accepting chemotaxis proteins（MCP）plays an important role in signaling and epiphytic infection,so MCPs are worthy of studying.My experiment focus on identifying how nmcp genes work in Xoo and finding the key gene for signaling and epiphytic infection to lay the foundation of a further research in Xoo’s chemotaxis system.My experiment finds 10 candidate MCP genes and a doubtful one through bioinformatics methods,using double exchange method to study these genes’function.The results are as follows:（1）Genome-wide searching for mcp genes.MA domain is a typical protein domain in MCP,so this experiment scanned the coding region of MA domain at the level of whole genome,and 10 candidate mcp genes were identified.They are PXO₀0036,PXO₀0039,PXO₀0041,PXO₀0043,PXO₀0045,PXO₀0046,PXO₀0047,PXO₀0054,PXO₀6142 and PXO₀4751.PXO₀0916 gene,previously annotated as mcp gene of PX099A in GenBank,was excluded in list of mcp genes in this study,owing to the absent of MA domain in its predicted protein.（2）Construction of mcp mutants and MCP free strain.Designing primers,constructing mutants through the method of triparetal conjugation or electroporation transformation after cloning target sequence to plasmid pK18mobsacB,after resistance screening and PCR validation,the 10 deletion mutants and a MCP free strain of PX099A were obtained.Then,the chemotaxis genes were linked to the plasmid pXUK by cloning methods to construct complementary vectors,and then the corresponding complementary strains were obtained.Then complementary vectors were transduced into the MCP free strain one by one to observe the phenotype and pathogenicity of these strains.And It was verified that the chemotaxis gene PXO₀6142 plays a key role during the course of the signaling and epiphytic infection of PX099A.（3）The basic phenotypes of all mutants were detected.The results showed that the swimming ability of mutants with PXO₀0041 and PXO₀0045 decreased slightly,and that of deleted mutants with PXO 06142 and MCP free strains decreased significantly.The deletion mutants of PXO₀4751,PXO 06142 and MCP free strains showed significantly lower mobility.The osmotic resistance of PXO₀4751 deleted mutant was weakened.（4）The virulence of all mutants was tested.It was found that there was no significant difference in virulence between the mutants and wild type in the case of leaf cutting inoculation.In order to test the variation of virulence of the mutants and MCP free strains more accurately,then the non-traumatic spray inoculation method was used to detect the pathogenicity changes of the mutants and MCP free strains.The results showed that the pathogenicity of PXO₀6142 deletion mutant and MCP free strain was significantly lower than that of wild type in spray inoculation experiment.And the virulence of PXO 06142 deletion mutant and MCP free strain can be complemented.In summary,after constructing a single gene deletion mutant,we found that gene PXO₀6142 played an important role in rice epiphytic infection.Therefore,we further constructed a MCP free strain and conduced each gene into MCP free strain one by one,then tested their phenotype and pathogenicity,which verified the key role of gene PXO₀6142.It is concluded that the chemotaxis genes of PX099A plays an important role in the early colonization and infection.Among all the chemotactic genes,the gene PXO 06142 plays an important role in phenotype and pathogenicity.