Bioinformatic Analysis And Expression Study Of Pathogenicity-related Genes Involved In Fusarium And Host Interaction

In this thesis,the morphological taxonomy and molecular biological measures were employed to clarify the main pathogens of tomato Fusarium wilt in Shanxi and their genetic differentiation.Based on the bioinformatics,the prtotein interaction networks of different pathogenic factors of Fusarium oxysporum f.sp.lycopersici were constructed through STRING protein interaction network database and Cytoscape software,the regulatory mechanisms and biological functions of pathogenicity-related genes involved in each protein interaction network were further analyzed with DAVID online analysis tool.The expression of hub genes,pathogenicity-related genes,pri-miRNAs,milRNAs' target genes of F.oxysporum f.sp.lycopersici and the pathogenesis-related genes was evaluated by RT-qPCR to further elucidate their molecular function and regulatory mechanism.In addition,the sub-culturing effectiveness of F.graminearum and F.proliferitum were studied by morphological observation,pathogenicity identification and ISSR molecular markers to clarify the genetic variation of wild strains under subcultural conditions.The major results are summarized as follows:1.A total of four pathogens were identified as of tomato Fusarium wilt,which were Fusarium oxysporum,Fusarium solani,Fusarium verticillium and Fusarium moniliforme var.subglutinans.Among them,the isolating frequency of Fusarium oxysporum was 53.3%,significantly higher than that of other strains,which was the main pathogen.It is the first report of Fusarium oniliforme var.subglutinans that causing tomato Fusarium wilt in the world.2.Of the fifty-six F.oxysporum strains isolated from diseased tomato plants,three physiological races of F.oxysporum f.sp.lycopersici were identified,which were physiological race 1,2,and 3.The results showed that the race 1 is dominant pathogen in Fusarium wilt diseased tomato plants in Shanxi.It is the first report of physiological race 3 that causing tomato Fusarium wilt in China.3.The genetic diversity of 56 strains of F.oxysporum showed that the selected primers could distinguish the F.oxysporum f.sp.lycopersici from the rest forma specialis of Fusarium oxysporum,but could not clearly distinguish the physiological races of F.oxysporum f.sp.lycopersici.4.The protein interaction networks of pathogenicity genes,carbohydrate active enzyme,transcription factor,lipase,ABC transporter,and G-couple protein receptor showed that the interaction levels of proteins in each protein interaction network were different.The protein interaction of the G-coupled protein receptor was relatively close.In each protein interaction network,the interaction levels of modules were also different.The biological processes,molecular functions and cell components of kernel modules in each protein interaction network directly reflected the biological functions involved in the biological activity.It is found that some genes involved in the common biological processes appear in the several protein interaction networks which reflecting the biological process of organisms as a unified whole that requires the participation of common genes.5.The protein interaction network module of hub genes was constructed by STRING protein interaction network database and Cytoscape software.The gene enrichment analysis by DAVID tools showed that the selected core genes are mainly involved in cell signal transduction.6.The results of gene expression of the pathogenicity-related genes in F.oxysporum f.sp.lycopersici and the pathogenesis genes in tomato during different period of pathogen-host interaction showed that three genes?FOXG₁3835,FOXG₀4162 and FOXG₀6022?were involved in the pathogen self-defense in the early stage of the host-pathogen interactions;FOXG₀8410,FOXG₀8140,FOXG₀4346,FOXG₀8598,FOXG₀3382,FOXG₀8517 and FOXG₁2803 were closely related to the pathogen primary infection and colonization;FOXG₀0154 and FOXG₀0893 were correlated with pathogen expansion in infected tomato plant;NM₀01288651,XM₀04238172,XM₀04245506 and AW154864.1 genes of tomato were involved in host resistant reaction to pathogen.7.The protein interaction network of milRNAs and their target genes of F.oxysporum f.sp.lycopersici showed that the interaction between milRNA₁,milRNA₂,milRNA₃ and milRNA₈ were more closely,and the target genes FOXG₀4240 and FOXG₀3470 could interact with many genes and could be regarded as the hub gene of the interaction network.The main molecular function and biological process of the protein interaction network was oxidordeuctase activity and the small GTPase mediated signal transduction.8.The gene expression levels of pri-milRNAs and their target genes during pathogen-host interaction showed that the tested pri-fox_milRNA₂a,pri-fox_milRNA₂b and FOXG₀3470,fox_milRNA₃b and FOXG₀4910,fox_milRNA₄ and FOXG₀8812,fox_milRNA₆ and FOXG₀0067 were negatively regulated.FOXG₀3470 and FOXG₀4910 play a part in initial stage of pathogen infection.9.Subculturing strains of Fusarium graminearum showed significant phenomena of degradation,such as the decreasing of mycelial growth rate,the increasing of sporulation amount,and the decreasing of pathogenicity,wheres,the subculturing straisn of Fusarium proliferitum did not show significant phenomena of degradation,such as the slight decreasing of mycelial growth rate,the unnoticeable decreasing of pathogenicity,and decreasing of sporulation amount.The genetic diversity of subculturing strains of Fusarium graminearum and Fusarium proliferitum showed that the genetic differentiation of the two species was observed significantly;However,the genetic differentiation and the diferential biological characteristics of the two species could not by ISSR molecular marker.