Functional Characterization Of FGSG₀7836 Gene In Fusarium Graminearum

Fusarium head blight is one of the major diseases of wheat and is prevalent on a global scale.The main pathogen causing Fusarium head blight is Fusarium graminearum.At present,the sequencing of the genome of Fusarium graminearum has been completed.It is very important for studing the pathogenic mechanism of genes associated with the virulence of the pathogen of Fusarium head blight effectively.The DGE sequencing analysis for MGV1 knock-out mutant was carried out before this study.There were 1729 up-regulated genes and 2062 down-regulated genes to be found in MGV1 knock-out mutant compared with the wild type strain of Fusarium graminearum.The gene differential expression analysis showed that the expression of FGSG₀7836 gene was significantly decreased after MGV1 gene deletion,suggesting that the FGSG₀7836 gene was one of the down stream elements in MGV1 pathway.Go annotation given out that the FGSG₀7836 gene was involved in the ATP binding and protein dimerization activity.In order to study the function of the gene,the gene knock-out strategy was applied in this study.To delete FGSG₀7836 gene,a knock-out cassette containing the hygromycin resistance gene hph was constructed by Split-Marker technique,the cassette was transformed into the protoplasts of PH-1 strain mediated by PEG solution.The transformants were selected on the medium containing hygromycin and screened by PCR to identify deletion mutants and then to carry out the phenotypic and pathogenic assay for the deletion mutant.To make the complementation for the deletion mutant,the full sequence of FGSG₀7836 gene was amplified by primer FGSG₀7836 com1 F / FGSG₀7836 com2 R and was cloned into the BamHI site of p ZWH1 and finally a complementary vector was obtained,nominated p ZPH1.The complementary vectors were transformed into the deletion mutant by PEG-mediated method and transformants were selected by G418.The recovery mutants were confirmed by PCR.The results showed that there were no significant difference between mutant and wild type PH-1 of Fusarium graminearum on the colony morphology,conidial morphology,pathogenicity and the filtrate colour of the medium after culture.However,the △ FGSG₀7836 grew greatly slow compared with the wildtype.The asexual and sexual assay showed that the amount of conidia the △ FGSG₀7836 decreased signifantly and the perithecia produced less greatly than that of the wild type.After digestion by the enzyme,the protoplast of the △ FGSG₀7836 released faster than that of the PH-1.When cultured at high temperature,the swollen top of the mycelium and hyphae fracture were observed in △ FGSG₀7836 but not in PH-1.The result of digestion and the swollen top of the hyphae suggested that the cell wall of △ FGSG₀7836 was weak,indictive that FGSG₀7836 is a gene related with the cell wall integration in Fusarium graminearum.The complementation experiment demonstrated that the complementation mutant was able to recover in colony morphology and other phenotype of PH-1,confirmed that the phenotypic changes of the knockout mutant were indeed caused by the deletion of the FGSG₀7836 gene.