| Trichoderma spp.,which is widespread and effective biocontrol agent,has huge potential and widely used in agriculture and forestry.Under the bio-control mechanism studying,Trichoderma can not only inhibit pathogenic fungi,but also directly affect host plants and stimulate host plants to produce systemic resistance to pathogenic fungi.Xylanase secreted by.asperellum belongs to Glycosyl Hydrolase Family 11(GH11),which can stimulate host plants to produce systemic resistance to pathogenic fungi.It is called a new plant inducer and has high scientific research value and broad development prospects.In this study,there were four Xyn genes in the GH11 family of T.asperellum ACCC30536.Xyn29.4 has one intron,282 amino acids(aa),isoelectric point(pI)5.51,protein molecular weight(MW)29.36 kDa;Xyn24.2 has one intron,223 aa,pI 6.82,MW 24.20 kDa;Xyn24.4 has one intron,230 aa,pI 5.00,MW 24.41 kDa;Xyn24.1 has two introns,225 aa,pI 5.25,MW 24.08 kDa.The four Xyn genes of T.asperellum GH11 family can be classified into four groups:?,?,? and ? respectively.The gene structure and conserved domain distribution of each group are highly similar.According to the analysis of gene structure,Trichoderma spp.,Aspergillus fumigatus and Aspergillus nidulans have the same conservative motif2,motif6 and motif8.It is speculated that the mechanism of Xyn hydrolysis is similar between them.Compared with Xyn genes of A.fumigatus and A.nestii,Xyn genes of Trichoderma spp.contains more conservative motifs and is more closely arranged,which indicates that Xyn genes of Trichoderma spp.may have higher accuracy when it binds to substrates.At the protein level,the characteristic semi-folded thumb region of GH11 family xylanases is very conservative,and thumb-like rings are absolutely necessary for their hydrolytic activity.Xyn29.4,Xyn24.4 and Xyn24.1 belong to type ? endo-1,4-beta-xylanase and Xyn24.2 belong to type ? endo-1,4-beta-xylanase.This study provides a method to treat conidia of Trichoderma sp.,which is easier to extract RNA from the treated conidia of Trichoderma sp..The transcription level of xylanase genes of.asperellum GH11 family was determined by RT-qPCR in the co-culture of T.asperellum ACCC30536 conidia and Populus davidiana ×.alba var.pyramidalis Louche(PdPap)seedlings.Under the condition of co-culture of.asperellum conidia and PdPap seedlings,Xyn24.4 and Xyn24.1 genes of T.asperellum strains were not expressed under this condition.The expression pattern of type I endo-1,4-beta-xylanase gene(Xyn29.4 gene)of.asperellum strains was similar to that of type II endo-1,4-beta-xylanase gene(Xyn24.2 gene),and their expression peaked at 12 hours.Xyn gene of T asperellum GH11 family plays an important role in the interaction between T.asperell T.strains and PdPap seedlings.To study characteristics of xylanase from T.asperell T.GH11 family and mechanism of inducing plant systemic disease resistance,the expression vectors pPIC9K-Xyn29.4 and pPIC9K-Xyn24.2 were successfully constructed.Xylanase yeast engineering strains GS115-Xyn29.4 and GS115-Xyn24.2 were successfully constructed.Xylanase yeast engineering strains GS115-Xyn29.4 and GS115-Xyn24.2 successfully expressed xylanases:29 kDa type ?recombinant xylanase and 24 kDa type ? recombinant xylanase.The highest activity of type ?recombinant xylanase and type ? recombinant xylanase were 18.9 IU/mL and 20.4 IU/mL,respectively.The recombinant xylanase rXyn29.4 and rXyn24.2 were obtained.In order to study mechanism of xylanase inducing disease resistance in woody plants,the gene transcription patterns and physiological changes of PdPap seedlings induced by recombinant xylanases were discussed.(1)Recombinant xylanases induces positive regulation of JAR1 gene and negative regulation of COI gene,while JAZ transcription level increases first and then decreases,which activates ORCA3 expression.Recombinant xylanase can stimulate jasmonic acid signal of PdPap seedlings,thus establishing induction system resistance(ISR)of PdPap seedlings,which makes PdPap seedlings broad-spectrum resistance to pathogens.(2)The above-ground fresh weight,dry weight,plant height,leaf length,leaf width,root length and root number of PdPap seedlings induced by recombinant xylanase were significantly higher than those of non-induced PdPap seedlings.Recombinant xylanase can control the expression of auxin signal related genes in PdPap seedlings,and ultimately activate GH3 expression to regulate plant growth.Therefore,it can be concluded that recombinant xylanase can promote the growth of PdPap seedlings.(3)Recombinant xylanase enhances the catalase(CAT)activity of PdPap seedlings leaves,thus reducing the accumulation of hydrogen peroxide,at the same time reducing the content of malondialdehyde(MDA),further improving the antioxidant capacity of PdPap seedlings.In the staining experiments of PdPap seedlings induced by recombinant xylanase,Evans Blus and NITROBLUE TETRAZOLIUM(NBT),the area of leaf cell necrosis was reduced,which again showed that recombinant xylanase increased the defense enzyme activity of PdPap seedlings and decreased the accumulation of reactive oxygen.In order to study the mechanism of xylanase inducing disease resistance to woody plants,the resistance ability and resistance pathway of PdPap seedlings induced by recombinant xylanase were discussed.In vitro leaf experiments of PdPap seedlings,the stomatal shape of leaves inoculated with Alternaria alternata after recombinant xylanase induction and the stomatal shape and opening degree of healthy leaves were basically the same.The smaller incidence area,stomatal shape and opening degree of A.alternata in leaves of PdPap seedlings showed that recombinant xylanase significantly induced systemic disease resistance of PdPap seedlings,which was good for prevention to A.alternata infection.In the experiment of inoculating Rhizoctonia solani and Fusarium oxysporum into the whole plant of PdPap seedlings,the whole plant of PdPap seedlings in the treatment group of recombinant xylanase induction followed by R.solani and.oxysporum infection showed no obvious signs of natural disease,the underground roots were very healthy,and the pre-induction of recombinant xylanase could activate resistance of PdPap seedlings to R.solani and F.oxysporum.The RT-PCR data showed that jasmonic acid signaling pathway of PdPap seedlings was regulated after recombinant xylanase induction,and the experimental data of jasmonic acid in leaves of PdPap seedlings after recombinant xylanase induction showed that the jasmonic acid content in leaves of PdPap seedlings changed greatly with time,especially at 12 hours,the jasmonic acid content in leaves of PdPap seedlings reached 2.2 of that of control group.Recombinant xylanase could stimulate jasmonic acid content of PdPap seedlings to increase first and then decrease.In the experiment of local or systematic induction of disease resistance of PdPap seedlings by recombinant xylanase,root induction of recombinant xylanase can induce systemic disease resistance of PdPap seedlings.Moreover,only local root induction of PdPap seedlings can stimulate the whole plant to produce systemic disease resistance.The leaves of PdPap seedlings were sprayed with methyl jasmonate for induction,and then the left roots of the experimental group infected by F.oxysporum did not show obvious incidence.Recombinant xylanase stimulated PdPap seedlings to produce more jasmonic acid,thereby inducing disease resistance of PdPap seedlings system.In summary,cloning,bioinformatics analysis and transcription pattern of Xyn29.4 and Xyn24.2 genes of T.asperellum ACCC30536 strain GH11 family provide basic data for the study of recombinant xylanase inducer;the eukaryotic expression and characterization of recombinant xylanase rXyn29.4 and rXyn24.2 provide theory for the development and utilization of new plant inducers basis and technical support;xylanase rXyn29.4 and xylanase rXyn24.2 can promote the growth of PdPap seedlings,and more importantly,they can induce PdPap seedlings from local roots.Inducing of recombinant xylanase to PdPap seedlings through jasmonic acid pathway can induce disease resistance of PdPap seedlings system,which provides theoretical support for the study of the mechanism of inducing plant disease resistance by recombinant xylanase. |
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