Colonization Dynamics And Extracellular Polysaccharide Structure Identification Of Rhodopseudomonas Palustris And Induced Resistance Mechanism

Rhodopseudomonas palustris,is widely used in the files of food industy,animal husbandry,environmental governance and agriculture,due to its high content of biologically active co-factors.Microbial exopolysaccharide(EPS),as a kind of natural macromolecular compound,is widely found in bacteria,fungi and some algae.Because of its antioxidant,antiviral,anti-tumor and immune-enhancing functions,which has a broad potential development in various of fields.In previous report,we had illuminated that the R.palustris GJ-22 can promote plant growth,and induce plant resistance to tobacco mosaic virus(TMV)by producing two plant hormones,IAA and ALA.In this study,R.palustris GJ-22 was selected as the research object,gfp-tagged strain GJ-22-gfp was constructed to observe colonization dynamics in tobacco phyllospheric.We constructed a mutant of the exopolysaccharide biosynthes is gene and found that the yield of exopolysaccharide was correlated with the colonization dynamics of the strain GJ-22.In this study,an extracellular polysaccharide was extracted from the fermentation broth of R.palustris GJ-22,its structure was characterized and analyzed,and the mechanism of EPS induced plant systemic resistance was further explored,which provided more theoretical basis for the development and application of EPS.The specific research results were as follows:1.Construction of gfp-tagged strainDue to the difficulty in construction of fluorescence-labeled strains of R.palustris,the method of homologous recombination was used in this study.The homologous arm genes of R.palustris and gene gfp were linked to the final vector p BBR1MCS-2 by the method of enzyme restriction and ligation successively,and then the recombinant vector p BBR1-pck A-gfp was constructed.Finally,the recombinant vector was transformed into competent cells of R.palustris GJ-22 by electric transformation,and the gfp-labeled strain GJ-22-gfp was constructed successfully.In addition,the expression stability of fluorescence,growth curves and the biocontrol function of the labeled strain were verified.Compared with the wild-type strain,the differences were not significant,indicating that the gfp-labeled strain GJ-22-gfp could meet the needs of the next experiments.2.Observationthe colonization dynamics of R.palustris GJ-22 in tobacco leaf and identification its influencing factorsConfocal laser fluorescence microscopy(CLSM)and scanning electron microscopy(SEM)were used to observe the colonization dynamics of strain GJ-22-gfp in the leaf of tobacco.The results showed that there were four stages in colonization establishment.Stage I:In the early stage of colonization,most of the bacteria were scattered on the surface of leaves with the form of a single strain.In stage II:the number of bacteria decreased dramatically.The bacteria formed small bacterial communities and lived at the junction of leaf epidermal cells.Stage III:The number of bacteria tends to be stable.The bacteria gather ed together,and formed small bacterial aggregates.Stage IV:Bacterial colonization is in the mature stage,the number of bacteria increased slowly,small bacterial aggregates grow into large bacterial aggregates,and spread around.Through the detection of induced resistance of plants at different stages,it was shown that when the bacteria colonized in the stage III or stage IV,it could enhance plant induced resistance,and improve plant immunity.In addition,the extracellular polysaccharide content of exocellular polysaccharide synthesis gene mutant strains?Exop1 and?Exop2 were determined.Results found that the mutant strain?Exop1 produced decreased extracellular polysaccharide,and the number of colonization reduce significantly,the induce plant resistance was also reduced.In a word,these results indicated that extracellular polysaccharides(EPS)play an important role in colonization,and thus affect the induced resistance in tobacco.3.Isolation,purification and structure identification of exopolysaccharides from Rhodopseudomonas palustrisAn extracellular polysaccharide(EPS)was extracted from the fermentation broth of strain GJ-22 by centrifugation and alcohol precipitation.A single component of pure polysaccharide G-EPS was obtained after purification.The molecular weight of exopolysaccharides were determined by high performance gel penet ration chromatograph(HPGPC).The results showed that the molecular weight of G-EPS was10.026 k D,and the FT-IR spectra showed that G-EPS was?-glycoside bond configuration.The polysaccharides were hydrolyzed and derivatized,and then the monosaccharide compositions were analyzed by GC-MS.The results showed that the exopolysaccharides were composed of mannose and glucose monosaccharide units,with a ratio of 116:9.Together with the datas of methylation,1 D NMR and 2D NMR,result showed that G-EPS was linked to mannose by(1?)linked mannopyranose,(1?2)linked mannopyranose,(1?3)linked mannopyranose,(1?6)linked mannopyranose and(1?2,6)linked mannopyranose,and the molar ratios were 3.1:2.0:0.8:0.6:3.5.Finally,the molecular structure formula of G-EPS was deduced,which provided an effective theoretical basis for the future study of structure-activity relationship and structure optimization of extracellular polysaccharides.4.Transcriptome analysis of the mechanism of exopolysaccharide induce d systemic resistance in tobaccoThe differences in transcriptional regulation between dd H₂O and extracellular polysaccharide(EPS)were explored in N.benthamiana.Results showed that there were 6049 differentially expressed genes between the treatment s,among with 2283were up-regulated genes and 3766 were down-regulated genes.Combined with KEGG enrichment analysis and q PCR detection of significantly expressed genes,indicated that G-EPS induced plant immune response might through SA,JA/ET and MAPK pathway.5.Exopolysaccharide G-EPS induced defense enzyme activity in N.benthamiana to enhance immune defense responseThe promotion and induced systemic resistance to TMV in plant of exopolysaccharide G-EPS were investigated.The results showed that the exopolysaccharide G-EPS could promote plant growth significantly.Compared with the control treatment,the root length and dry weight of plants treated with G-EPS increased by 26.55%and 37.1%,respectively.In addition,the induced resistance to TMV of tobacco treated with G-EPS was enhanced significantly.At the same time,the changes of the activities of anti-disease related defense enzymes in leaves of tobacco plants treated with G-EPS were also investigated.The results showed that G-EPS could enhance the activities of POD,PAL and SOD in plants significantly,so as the contents of hydrogen peroxide and chlorophyll in plants,so it could promote the synthesis of plant protectins and lignin,so as to improve th e resistance of plants to TMV.In addition,G-EPS treatment reduced the content of malondialdehyde(MDA)in plants,and reduced the damage of virus to plant cell membrane,so as to protect plants and enhance plant resistance.