| Tobacco mosaic virus(TMV)as one of the most destructive plant viruses causes serious economic losses in most agricultural areas.Biopesticides have attracted more and more attention and showed good development potential.Isolation and identification of antiviral substances from beneficial microorganisms in nature has become a research hotspot.In this study,we screened the multifunctional biocontrol bacteria with antiviral activity on TMV and growth promoting effect on plants,isolated and purified the antiviral substances,cloned its genes,obtained the purified recombinant protein through prokaryotic expression.The anti-TMV mechanism of antiviral substances was analyzed both from inducing resistance to host and inactivation.The main results of this study are as follows:1.The multifunctional biocontrol bacteria B8 was screened to antiviral activity and promote plant growth.In this experiment,8 strains of biocontrol bacteria with inhibitory effect on Bacillus cereus were screened out from 23 strains of bacteria by “cylinder plate method”.We researched the inhibitory effect of 7 strains of fermentation fluid both on Bacillus cereus and TMV,and the effect of promoting tomato growth.The results showed that the fermentation broth of strain B8 could effectively inhibit Bacillus cereus.After the mixture of the fermentation broth and TMV in vitro,the inhibition rate of necrosis was 87.52% on Nicotiana glutinosa.The protective and curative effects of fermentation broth on TMV were 62.40% and 58.91%,respectively.The crude extract of antagonistic protein B8 was mixed with TMV and inoculated with Nicotiana glutinosa and the inhibition rate was 42.50%.When tomato seeds were treated with 20 times diluted fermentation liquid,the growth of tomato seedlings could be promoted.2.Isolation and identification of antiviral protein of Brevibacillus laterosporus B8.In this experiment,the components with inhibition on TMV infection were separated by ammonium sulfate precipitation,thin layer chromatography,Sephadex LH-20 column chromatography high performance liquid chromatography.The molecular weight of the protein was about 12.8 k Da.Mass spectrometry showed that the protein was a protein with unknown function.We named it BLB8.Further analysis showed that the region of BLB8 signal peptide was predicted to be 1-30 amino acids.Antiviral protein BLB8 can induce hypersensitivity response(HR)in tobacco.3.Gene cloning,prokaryotic expression and purification of recombinant protein BLB8 were studied.According to the results of purified BLB8 mass spectrometry,the coding gene blb8 was cloned from Brevibacillus laterosporus B8.And the prokaryotic expression vector p Cold-SUMO-blb8 was successfully constructed.The expression of SUMO-BLB8 fusion protein was induced by IPTG in Escherichia coli.The recombinant protein BLB8 was obtained by enzymatic digestion of SUMO-BLB8 fusion protein with SUMO protease.4.The mechanism of antiviral protein BLB8 inhibiting TMV was studied.In this study,the activity and mechanism of recombinant BLB8 resistance to TMV were verified by many methods.The results of half leaf method showed that the inactivation effect of TMV on Nicotiana glutinosa reached up to 83% when the concentration of recombinant BLB8 was 200 ?g·m L-1.The protective and curative effect of BLB8 against TMV were 63.83% and 55.21%,respectively.The inhibition rate of transient expression of BLB8 to TMV in tobacco leaves was about 76%.The infiltration experiment showed that BLB8 could effectively inhibit the multiplication and extension of TMV in tobacco leaves and cause systemic resistance to TMV infection.The results of RT-q PCR showed that the transcription level of TMV-CP was three times lower than that of the control.And the new leaves at the top showed only slight malformation.Subcellular localization assay indicated that BLB8 and TMV-GFP co-localize in plasma membrane in tobacco.And transmission electron microscopy(TEM)observations discovered that TMV particles was destructed greatly after incubation with BLB8 but not BSA.5.The mechanism of inducing resistance to TMV in tobacco by antiviral protein BLB8 was studied.The results showed that both natural and recombinant BLB8 could induce HR response of tobacco.The HR marker gene HSR203 J in BLB8 infiltrating leaves was induced and also induced the production of reactive oxygen species(ROS)signal,and the lowest concentration of HR induced by recombinant BLB8 in tobacco leaves was 100 ?g·m L-1.The expression of defense related genes in BLB8-treated tobacco was detected by RT-q PCR.The results showed that BLB8 treatment resulted induced the expression of defense-related genes including PR1 a,PR5,NPR1,PAL,PDF1.2 and COI1.The results showed that BLB8 mainly triggered the immune response by activating SA signaling pathway.A total of 242 differential expression genes(DEGs)were obtained by transcriptome analysis,of which 231 genes were up-regulated and 11 genes down regulated.In the BLB8-induced defense response,plant response to adversity,defense response and related genes of plant pathogen interaction wereup-regulated.These genes are involved in hormone signaling,transcription factors and defense response genes.According to the analysis of Go enrichment,the molecular functions of DEGs in BLB8 treatment group were mainly concentrated in catalytic activity,binding,metabolism and cell treatment.In addition,52 DEGs were annotated to 20 different KEGG pathways.We speculated that BLB8 could induce resistance to TMV through SA signaling pathway. |
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