| The banana fusarium wilt,which is caused by Fusarium oxysporum f.sp.cubense(Foc),is one of the most devastating diseases of banana.Especially,Foc4 is the most severe disease.According to the previous secreted proteome research in this laboratory,it was found that the expression of polyamine oxidase(PAO)in Foc4 was significantly up-regulated after treatment with banana root extract.It was speculated that PAO may be involved in the pathogenesis of Foc4.At present,the gene function of polyamine oxidase in Foc4 has not been reported.In this research,the gene function of polyamine oxidase in Foc4 was studied for the first time by methods such as gene knockout,gene complementation,phenotypic observation,and pathogenicity analysis.The specific results are as follows:(1)The expression of polyamine oxidase gene(Fo Pao)in Foc4 was analyzed by q RT-PCR.The results showed that the expression of Fo Pao in Foc4 was significantly up-regulated after being induced by banana root tissue,which was consistent with the results of secreted proteome in this laboratory.(2)The bioinformatics method was used to analyze the polyamine oxidase in Foc4.The results showed that after analysis by Signal P 4.1 Server,Wo LF PSORT,and TMHMM Server v2.0,it was found that the polyamine oxidase has an N-terminal signal peptide and the secretory pathway is unknown.Further analysis revealed that the polyamine oxidase gene has 1982 bp DNA sequence,527 amino acids,contains an amino oxidase(Amino oxidase)domain and a flavin adenine dinucleotide(FAD)binding site.Phylogenetic tree analysis found that polyamine oxidase is a highly homologous protein in many Fusarium and has good conservation;there is another gene encoding polyamine oxidase in Foc4,both of which have 66.8% in gene sequence similarity and 69.6% similarity in amino acid sequence.(3)A homologous recombination gene knockout strategy was used to successfully construct a knockout vector containing a hygromycin selection marker.Further,the PEG-mediated protoplast transformation method,PCR analysis,and Southern blot was used to identify the hygromycin resistance mutant.Three knockout mutants of the polyamine oxidase gene(ΔFo Pao)of Foc4 were obtained.A randomly inserted gene complementation strategy was used to successfully constructed a complementation vector containing a bleomycin selection marker.Four polyamine oxidase gene complementation mutants(ΔFo Pao-7-com)were obtained using by protoplast transformation.(4)The phenotypic analysis of ΔFo Pao showed that compared with Foc4 wild type,ΔFo Pao’s spore production and dry mycelium weight were significantly increased,but there were no significant difference in colony growth,conidial morphology,mycelial morphology,penetrating ability and colonization ability to apples;the results of the stress resistance test showed that for the ΔFo Pao and Foc4 wild-type,no significant difference in growth was observed on the PDA medium containing 1 mol/L Na Cl、1mol/L sorbitol and 10mmol/L Li Cl as compared to the growth on PDA alone;the results of its cell wall integrity test showed that the absence of Fo Pao did not affect the cell wall integrity of Foc4;The results of its tolerance to exogenous polyamines showed that the absence of Fo Pao did not affect the colony growth of Foc4 on PDA medium containing 5 mol/L spermidine;Compared with the Foc4 wild type,the results of the Pao inhibitor test showed that at low inhibitor concentration,the colony edge of ΔFo Pao was sparse,and at high inhibitor concentration,there was no significant difference in the colony of ΔFo Pao.(5)The relative biomass of ΔFo Pao in Brazil banana roots was analyzed.The results showed that at 24 h and 48 h after inoculation,the biomass of ΔFo Pao in Brazil banana roots was significantly higher than that of Foc4 wild type.(6)Analysis the pathogenicity of ΔFo Pao showed that after inoculation the ΔFo Pao,the yellowing area of the leaves and the browning area of bulbs were significantly higher than those of inoculation the Foc4 wild type;The disease index statistical results also show that the pathogenicity of ΔFo Pao is significantly higher than that of Foc4 wild type.Therefore,ΔFo Pao was more pathogenic than that of Foc4 wild type.(7)In order to further analyze the cause of its enhanced pathogenicity,we further analyzed the synthesis of fusaric acid in ΔFo Pao and the expression of genes related to the polyamine synthesis and metabolic pathways where PAO is located.The results showed that compared with Foc4 wild type,fusaric acid synthesis was significantly increased inΔFo Pao;q RT-RCR analysis of genes related to polyamine synthesis and metabolic pathways.The ornithine decarboxylase gene(Fo Odc)is involved in the synthesis of putrescine.The deletion of the Fo Pao gene caused the Fo Odc gene expression to be significantly up-regulated after being induced by banana root tissues;The spermidine synthase gene(Fo Spe)is involved in the synthesis of spermidine.The deletion of the Fo Pao gene caused the amount of Fo Spe gene expression is up-regulation amplitude decreased compared with that of Foc4 wild type after 24 h of induction.The spermidine N1-acetyltransferase gene(Fo Ssat)participates in the metabolic pathway of polyamines.The deletion of the Fo Pao gene caused the Fo Ssat gene no longer up-regulates expression after being induced.q RT-RCR Analysis of Lae A that encoding polyamine secondary metabolic regulator showed that the deletion of Fo Pao gene caused the expression of Lae A to be lower than that of Foc4 wild type,whether or not induced. |
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