Functional Analysis Of Valine-glycine Repeat Protein VgrG Gene In Lysobacter Capsici X2-3

Biocontrol bacteria can promote the growth and development of plants,produce a variety of metabolites,and then play an antagonistic or competitive role,inhibit or kill pathogenic bacteria.They are important resources for biological control of plant diseases.The colonization ability,antimicrobial activity,environmental tolerance and so on of plant rhizosphere growth-promoting bacteria are the prerequisites for their role.Lysobacter capsici X2-3,isolated from wheat rhizosphere soil,has obvious antagonistic activities against many plant pathogenic fungi and oomycetes,and has good biological control potential.However,there is no gene knockout technology suitable for strain X2-3 at present.In this study,the suicide vector was used to construct a directional gene knockout method for L.capsici X2-3strain and the gene VgrG was knocked out.The characteristics of the mutant strain in biological characteristics,competitiveness and environmental tolerance were analyzed,which laid a foundation for further exploring the function and application of X2-3.The results of this study mainly include the following aspects:1.Sequence Analysis and Functional Prediction of Gene LC1470According to the results of X2-3 genome sequencing and functional prediction,the gene LC1470 may encode a conserved protein of the Vgr family of VI secretory system.Compared with GenBank and Blastp,it discovers that LC1470 has 96.35%homology with VgrG genes inL.antibioticus and L.capsici,which is highly conservative.2.Obtainment of VgrG gene knockout mutant and gene complemented mutantOn the basis of gene sequence,specific primers were designed to amplify thehomologous arms of gene VgrG,and the homologous arms were connected to suicide vector pKMS1 to construct the pKMS1-VgrG knockout recombinant vector.The VgrG deletionmutants were preliminarily obtained by electroporation into strain X2-3 competent cells and screened by kanamycin(Km 500 ug.mL^-1)and 10%sucrose plate.M651 was confirmed to be a VgrG deletion mutant by PCR and Southern blot.In the light of genome,specific primers were designed to amplify the complete ORF of LC1470.After double digestion with Hind?and BamH?,the fragment was linked with the same double digestion host vector pBBR1-MCS5.The recombinant vector pBBR1-1470 was successfully constructed.The recombinant plasmid pBBR1-1470 was transformed into themutant M651,and the complementary strain MCS18 was obtained by phenotypic observation and PCR verification.3.Analysis of biological characteristics of wild and mutant strainsBy comparing the colony morphology of wild strain,mutant M651 and complementary strain MCS18,it was found that the surface of wild strain X2-3 was smooth and thick,the surface of mutant strain M651 was wrinkled and low in viscosity,while complementary strain MCS18 restored the morphology of wild strain.The growth rate of mutant strain M651 was significantly lower than that of wild strain X2-3 at all time points.The complementary strain MCS18 basically restored the level of wild strain,but there were significant differencesbetween the complementary strain MCS18 and wild strain at some time points.In view of the significant changes in colony morphology of mutant M651,the mutant was further analyzed in terms of biofilm,exopolysaccharide and motility ability.The results showed that the biofilm yield of wild strain X2-3 was prominently higher than that of mutant M651,and that of complementary strain MCS18 was lower than that of wild strain,but there was no remarkable difference between them.The exopolysaccharide production of mutant strain M651 was significantly higher than that of wild strain and complementary strainMCS18.The motility ability of mutant M651 was substantially lower than that of wild strain X2-3.The motility ability of the complementary strain MCS18 was basically restored to the level of wild strain,and there was no significant difference between mutant M651 and wild strain.Furthermore,the antimicrobial activities of wild strain X2-3,mutant strain M651 and complementary strain MCS18 was not dramatically changed by plate confrontation.In view of the significant changes in colony morphology of mutant M651,the mutant was further analyzed in terms of biofilm,exopolysaccharide and motility ability.The results showed that the biofilm yield of wild strain X2-3 was prominently higher than that of mutant M651,and that of complementary strain MCS18 was lower than that of wild strain,but there was no remarkable difference between them.The exopolysaccharide production of mutant strain M651 was significantly higher than that of wild strain and complementary strainMCS18.The motility ability of mutant M651 was substantially lower than that of wild strain X2-3.The motility ability of the complementary strain MCS18 was basically restored to the level of wild strain,and there was no significant difference between mutant M651 and wild strain.Furthermore,the antimicrobial activities of wild strain X2-3,mutant strain M651 and complementary strain MCS18 was not dramatically changed by plate confrontation.4.Stress Resistance Analysis of Wild-strains and MutantsBiofilm and motility ability are very important for bacterial life activities.In view of the significant changes in biofilm and sport ability of mutant strain M651,the tolerance of strain M651 was analyzed in terms of temperature,SDS,acidity and alkalinity,salt concentration and ultraviolet ray.The results revealed that the tolerances of the mutant strain M651 to high and low temperature,acidity and alkalinity,salt concentration and SDS were prominentlylower than that of wild strain.The tolerances of the complementary strain MCS18 were also different from that of wild strain,but the differences were not noteworthy.The mutant strain M651,wild strain X2-3 and the complementary strain MCS18 were not sensitive to ultraviolet radiation.These results indicated that the gene VgrG not only affected the biologicalcharacteristics of the strain,but also was related to the environmental tolerance of the strains.5.Competition Analysis of Wild Strains,Mutants against Other BacteriaRalstonia solanacearum,Burkholderia cenocepacia D-7 and Escherichia coli S17-1were selected as targets to analyze the competitive abilities of the wild strain X2-3,the mutant M651 and the complementary strain MCS18.By plate counting and Real-time fluorescence quantitative PCR?qRT-PCR?,the differences of competitive ability between the mutant and wild strain were analyzed.The plate counting method showed that the competitive abilities of the mutant strain M651 to R.solanacearum,E.coli S17-1 and B.cenocepacia D-7 wereweakened in varying degrees compared with wild strain,while the competitive abilities of complementary strain MCS18 were basically restored to the level of wild strains.Further analysis by qRT-PCR showed that after co-culture with E.coli S17-1,B.cenocepacia D-7 and R.solanacearum,the concentration ratios of the mutant strain M651were 12.57%and 13.81%,10.68%and 18.27%,1.59%and 2.08%at 24 h and 48 h,respectively,which were lower than those of the wild strain X2-3 and the complementary strain MCS18.It indicated that the competition of the mutant M651 was weaker than that of wild strain X2-3 and complementary strain MCS18.