Comparison And Pathogenic Function Analysis Of Mads1 And Flo1 From Fusarium Oxysporum F. Sp.cubense Race 1 And Race4

The banana fusarium wilt is a kind of fascicular systemic disease caused by Fusarium oxysporum f.sp.cubense.Itresults in destructive extermination of banana production and brought serious economic loss.Race 1 of the pathogen(Foc1) infests “Gros Michel” but not Cavendish cultivars.Race 4(Foc4) infests Cavendish cultivars and all cultivars susceptible to race1.Due to the lack of effective prevention methods and disease resistant varieties. So, it is very important to improve the pathogenic mechanism of Banana Fusarium wilt by screening the disease related genes.Based on the comparative transcriptomic analysis of race 1 and race 4 of Fusariumoxysporum f.sp.cubense induced with three carbon sources.BLAST the transcriptomic data with PHI database,we find five genes which were associated withvirulence.The Five gene istranscription factorcomp11743( Mads1,PHI:1070), comp13556(Flo1,PHI: 468),HC-toxin biosynthesiscomp14892(PHI:157),G protein comp17731(PHI: 385),Chitin synthasecomp20887(PHI: 389).The expression changes of these 5 genes in Foc treated banana roots were obtained by real-time fluorescence quantitative PCR.comp11743(Mads1)andcomp13556(Flo1)are up-regulated very obvious.The expression ofcomp11743(Mads1)at 48 h and 96 h is least 400-fold increase compared with the expression at 0h,and even the biggest can reach 900 times.The expression of comp13556(Flo1) in 48 h and 96 h although the ratio of 0 h up to multiples of less than A, but the average is also more than 90 times, the maximum can reach nearly 400 times.Although the expression of comp14892 in the Foc treatment after banana root also has up, up the multiple is not far, far less thancomp11743(Mads1) and comp13556(Flo1), and comp17731 and comp20887 in the Foc treatment of banana root after the expression of the downward trend.When pathogens infect host,some genes will highly expressed.So, we prior choose these two genes comp11743(Mads1) andcomp13556(Flo1)for further research.The sequences ofcomp11743(Mads1)were analyzed to see the same between Foc1 and Foc4.The full DNA length of comp11743(Mads1) were 633 bp in Foc1 and Foc4,all of them have no introns.The open reading frames of them were 633 bp, which encode 210 amino acids.The twocomp11743(Mads1) genes have the same nucleotide sites.The homology of the comp11743(Mads1)geneswas 100% in the nucleotide, and 100% in the amino acid levels.The sequences ofcomp13556(Flo1)were analyzed to see the differences between Foc1 and Foc4.The full DNA length of comp13556(Flo1)were 1010 bp in Foc1 and Foc4, consisting of 1 introns and 2 exons.The open reading frames of them were 960 bp,which encode 310 amino acids.The twocomp13556(Flo1) genes have differences in 10 nucleotide sites,which lead to differences in 2amino acid sites.The homology of thecomp13556(Flo1) geneswas 99.1% in the nucleotide, and 99.4% in the amino acid levels.The comp11743(Mads1),comp13556(Flo1)amino acid sequence blastp found thatcomp11743(Mads1)has a MADS SRF like functional domains,belong to the MADS family of SRF-like(I) protein,so,we named it Mads1;comp13556(Flo1)has a function of PA14 domain,belonging to the family of PA14, and has 50% homologyto Flocculation protein,so,we named it Flo1.Theflankingsequencesof Foc1-Mads1,Foc4-Mads1,and Foc1-Flo1,Foc4-Flo1 were obtained by TAIL-PCR.The method of homologous recombination was used to construct the knockout vector according to the flanking sequence of the measured target gene.Using ATMT methodtransformation,successfullyobtainedthe Foc1 and Foc4-Mads1,Flo1 genedeletionmuta nt Foc1-?Mads1, Foc4-?Mads1 and Foc1-?Flo1,Foc4-?Flo1.The culture shape of Foc1-?Mads1 and Foc4-?Mads1 on the PDA mediumplate was consistent with the wild type Foc1 and Foc4. The growth rate of mycelium was consistent with the wild type Foc1 and Foc4, and there was no difference in amount of spore between the mutant and wild type.The culture shape of Foc1-?Flo1 and Foc4-?Flo1 on the PDA mediumplate was consistent with the wild type Foc1 and Foc4. The growth rate of mycelium was consistent with the wild type Foc1 and Foc4, and there was no difference in amount of spore between the mutant and wild type. Using root dip inoculation method,Foc1-?Mads1, Foc4-?Mads1,Foc1-?Flo1, Foc4-?Flo1 knockout mutant and wild-type Foc were inoculatedfour-leaf stageseedlings of banana,45 days after the observation results. The results showed that, these bananas inoculated Foc1-?Mads1, Foc4-?Mads1 and wild-type Foc1, Foc4 all have the typical symptoms of banana wilt.The pathogenicity of mutants not appear significantly weakenedbecause of knockout Mads1 gene, it showed that Mads1 gene is not the key virulence factor.The disease index of Foc1-?Flo1, Foc4- Flo1 inoculated banana is 51.66±0.83 and 53.33±2.2,about 50% lower compared to the wild type Foc1 and Foc4.It shows thatalthoughthe deletionof Flo1 gene does not affect the normal growth of Fusarium wilt of banana, it can affect the virulence.In summary,Mads1 and Flo1 were highly expressed at Foc infection,preliminary evidence show Flo1 may be associated with virulence of Foc,Mads1 should be accompanied by a role in the infection process, but after gene knockout,no apparent virulence the impact.Maybe MADS-Box family of functional redundancy, there is a plurality of functions the same genes, when knocking out one gene, its missing features will be complementary to other genes in the same family.Therefore,subsequent trials can be cloned more other genes in this family for further research.