Establishment of ATMT Fusarium oxysporum f.sp. cubense race 4 effective transformation system and screening of the T-DNA insertional mutants.

【Objective】Based on agrobacterium-mediated transformation(ATMT), Fusarium oxysporum f.sp.cubense race 4 effective transformation system was established and mutant database of pathogenic bacteria was constructed to provide molecular markers for mutant gene and further research on functional gene of Fusarium oxysporum f.sp.cubense.【Method】In single factor variable experiment, testing material, fungal spore concentration, agrobacterium induction final concentration, IM induction medium pH, co-culture medium, co-culture AS concentration and co-culture temperature were studied to find out the key factors influencing transformation efficiency. Through morphologic observation and pathogenicity tests, mutants were screened. 【Result】The optimal conditions were A. tumefaciens(AGL1) induction concentration OD600=0.8, transformation receptor of 1×106conidia/mL, co-culture at pH 5.4 and 25 ℃, co-culture AS concentration of 200 μmol/mL and co-culture medium of nitrocellulose membrane. Under the improved conditions, transformation efficiency was in the range of 800-900 transformants per 106conidia. 【 Conclusion 】 GFP was successfully inserted and expressed in pathogenic bacteria. T-DNA mutant database of pathogenic bacteria was built. Many pathogenicity and phenotype changing mutant was screened to provide references for Fusarium oxysporum f.sp.cubense genome function research.