The Construction Of Genetic Transformation Method In Xanthomonas Citri Subsp. Malvacearum And Functional Analysis Of Hpaxm

Xanthomonas citri subsp.malvacearum(Xcm)can cause the cotton angular leaf spot,since the restriction-modification systems may exist in Xcm,it is rather difficult for the transformation of the exogenous genes,thus the genetic transformation of Xcm is still not easy.In this study,we broke through the barrier limiting the successful electroporation of Xcm and established an efficient gene deleted system which was suitable for Xcm,taking use of domesticating the wild-type strain or recombinant plasmids,this provides a scientific basis for future researchs on the functions of unknown genes in Xcm and other bacterias with the difficulties of genetic transformation.As a kind of hrp gene encoding HpaXm protein,hpaXm is amplified from the Xcm genome in our laboratory,and through the signal peptide prediction software,we predicted the first 45 bp of hpaXm may be a potential silimar signal peptide sequence,naming as hpaXm-IP.In this study,we took the eigth race of Xcm(V8)as the target strains,hpaKm and hpaXm-IP as the target genes,in order to verify the genetic transformation system of Xcm to be successful or not.I the knockout vectors were constructed successfullyWe amplified homologous arms of two target genes from V8 genome by PCR,the pK18mobsacB::N1012 and pK18mobsacB::m762 were constructed using for knockout hpaXm-IP and hpaXm through the fusion PCR.Results showed:pK18mobsacB::N1012 could be cut out a single band of approximately 1000 bp by double digestion,the same as homology arm hpaXm-IP-(L+R)of hpaKm-IP basically;basing on the comparison of sequencing results in the NCBI,nucleotide sequence was consistent hpaXm-IP-(L+R)completely,no base mismatching,it explained that pK18mobsacB::N1012 had been built successfully.PK18mobsacB::m762 could be cut out a single band of approximately 800 bp by double digestion,the same as homology arm hpaXm-(L+R)of hpaXm basically;basing on the comparison of sequencing results in the NCBI,nucleotide sequence was consistent hpaXm-(L+R)completely,no base mismatching,it explained that PK18mobsacB::m762 had been built successfully.II the strain and plasmids were domesticated successfullyStrain domesticating:when the screening concentration of azacytidine was increasing continuously,we inoculated the V8 strain on NA tablets including azacytidine for domesticating every generation,and picked the single colony of each generation preparing competent cells for electroporating,until obtaining the strain V8 of anti-100μM azacytidine which the restrict-modification system was mutated,results showed:three to twenty monoclonal were obtained every transformation.Plasmids domesticatiing:through ultrasonic extraction,we collected cell extracellular material(CFEs)of V8 containing methylases,and made CFEs and recombinant plasmids react in S-adenosine methionine(SAM)by 37 ℃,ten hours,then taking use of the mixture being fromed of phenol:chloroform:isoamyl alcohol to extract the supernatant fluid,the recombinant plasmids of being domesticated were obtained through ethanol precipitation and dissolved in TE buffer,results showed:we could get one to five monoclonal every transformation.Verification of the mutant strains:when strain or plasmids were not domesticated,we did not get any monoclonal on tablets,after domesticating,there could grow monoclonal which was plump,uplift and yellow luster,initially speculated that recombinant plasmids had been transformed into V8 strain by electricity conversion.Using the amplification primers of fusion genes and there homologous arms for PCR and sequencing test,we found that:the monoclonals from pK18mobsacB::m762 had been transformed into V8 strain by electroporating,only a single band of about 800 bp could be amplified and hpaXm genes could’t be amplified,instead,wild strains not only amplified a single band of approximately 1000 bp,but also could amplify the target band of about 400 bp;sequencing showed that the purpose gene was missing in monoclonal completely,we had got mutated bacteria of deleting hpaXm,naming as V8 Δ hpaXm.The monoclonals from transformed pK18mobsacB::N1012 into V8 strain,the results of the PCR and sequencing showed that the gene of 45 bp was also missing completely,that is to say,the silimar signal peptide sequence had been deleted,naming as V8ΔhpaXm-IP.Ⅲ Exploring the optimal conditions of electroporationAlthough genetic transformation of V8 strain had come ture through domestication successfully,and the efficiency of domesticating strain was ten to one hundred times as the domesticating plasmids,but the efficiency was still low.In order to make the mutated system of V8 strain more efficient,this experiment studied the factors that were likely to play roles in the efficiency when V8 strain carried electroporation one by one,such as:the growth condition of cells,voltage,concentration and dosage of plasmids,methods and time of recovering,type and temperature of recovery medium,the results showed:when we selected V8 in the late logarithmic for preparing competent cells,voltage 2.1 kv,7 μL plasmid as well as concentration of 50 μg/mL,recovery media SOC of 4 ℃ pre-cooling,culturing with time of four to five hours by oscillating,the efficiency of electrical conversion could reach about 5×102/μg DNA.IVthe biological characteristics of mutant strainsFrom the growth of tested strains on flats,we found that:the missing of hpaXm and hpaXm-IP did not affect the activity of extracellular polysaccharide,cellulose enzyme and extracellular lipase of V8 strain,the activity of extracellular amylase decreased;V8 strain itself didn’t have the ability to produce extracellular protease.Drawing the growth curve of all tested strains in NB,we found that the deletion of hpaXm and hpaXm-IP made V8 strain own the decreased ability to grow strain.Allergic reaction on tobacco showed that:the necrotic spots were formed on the leaves of tobacco by V8 strain,mutant strains V8 ΔhpaXm and V8 Δ hpaXm-IP,but the necrosis area formed by two mutant strainsbecame samller comparing with the wild strain,it speculated preliminary:the similar signal peptide sequence may have the ability to secrete traction of HpaXm protein,and HpaXm is not the only active material motivating HR in V8 strain;or the missing of hpaXm and hpaXm-IP play an influence on the pathogenic of V8 strain,but it is not necessary.