Optimization Of Agrobacterium Tumefaciens-mediated Transformation System And Screening Mutants In Aspergillus Flavus

Aspergillus flavus is infamous for the production of the most hepatotoxic and carcinogenic secondary metabolite known as aflatoxin B1.At present,the regulation mechanism of A.flavus producing toxin is still poorly understood,due to the lack of an efficient genetic research methods.In this study,an efficient Agrobacterium tumefaciens-mediated transformation for A.flavus was established,with a high-throughput screening method.A small-scale insertional mutant library was constructed.The main results were as follows:1.The transformation efficiency was increased when the mononuclear conidia were used as recipient materials.2.PgpdA initiates the expression of the resistance gene ble in A.flavus more efficiently than PtrpC promoter.3.A reliable and efficient genetic transformation system was established in this study.The optimized Agrobacterium-mediated transformation parameters for A.flavus is achieved using bacterial densities at OD₆₀₀=0.3,conidia numbers at 10⁶,Co-IM agar plate supplemented with 200?M acetosyringone,the co-culture of the mixture spores and bacterial cells was incubated at 22°C for 48h,and MM medium with 100?g/mL zeocin was used for selection.We successfully generated A.flavus transformants with an efficiency of⁶0 positive transformants per 10⁶ conidia using our protocol.Verification of positive transformants was performed via PCR and Southern blot.4.A high-throughput screening method for positive transformants was established.To screen and verify the positive transformants more efficiently,pDHB/G with the non-fused ble and gfp genes,pDHBG with the fused ble and gfp genes were constructed respectively.Fluorescence detection,in-gel fluorescence analysis,Western blot hybridization and genetic stability analysis showed that GFP could be normally and stably expressed in all transformants.The transformation efficiencies of the pDHBG was higher than that of the pDHB/G vector,which indicated that the fusion construct is more suitable for genetic transformation of A.flavus.5.Mutant phenotype screening and T-DNA insertion site analysis.A small-scale insertional mutant library?¹000 mutants?was constructed by T-DNA insertional mutagenesis.Several phenotypes of the mutants,including developmental charateristics?mycelial growth,colony color,conidiation?,aflatoxin B1 content and virulence against maize kernels were identified and characterized.And we isolated genonic flanking sequence of T-DNA insertions from some mutants,which will be beneficial for functional analysis of the pathogenic-related genes in A.flavus.