根癌农杆菌介导虎杖象甲培植梅花状青霉Penicillium herquei转化体系的构建OA

Agrobacterium tumefaciens-mediated transformation(ATMT)is widely used for genetic modification and functional analysis in filamentous fungi.This method exploits the process and transfer machinery of Agrobacterium tumefaciens to deliver T-DNA into the fungal genome through a cleavage and integration mechanism.As an obligate symbiont cultivated by attelabid weevils,Penicillium herquei plays essential roles in nutrient provision and ecological adaptation within the insect-fungus symbiotic system.However,the lack of a genetic manipulation tool has severely limited functional studies of this fungus.In this study,an efficient and stable ATMT system was established for P.herquei strain Ph506,a symbiotic fungus cultivated by the farming weevil Euops chinensis.Initially,the selection markers and their concentrations were determined through antibiotic sensitivity assays;75-100 μg/mL hygromycin B(Hyg)or 100-150 μg/mL geneticin(G418),combined with 200-300 μg/mL cefotaxime(Cefo),were used for transformant selection.Subsequently,shuttle plasmids carrying the mCherry red fluorescent protein gene and the corresponding resistance genes were introduced into Agrobacterium tumefaciens strain AGL-1 competent cells,respectively.Suspension of ground young fungal hyphae was used as the recipient and co-cultured with the Agrobacterium suspension on induction medium(IM)agar plates to facilitate T-DNA transfer.After 7 days of co-cultivation,the plates were overlaid with PDA medium containing the appropriate antibiotics to select resistant transformants.Genomic PCR confirmed the integration of the target gene into the genome,and fluorescence microscopy observation verified stable expression of the mCherry red fluorescent protein in the transformant hyphae.To further improve transformation efficiency,key parameters were systematically optimized using a one-factor-at-a-time approach.The optimal conditions were determined as follows:an acetosyringone(AS)concentration of 200 μmol/L,an Agrobacterium suspension density of OD600=0.6,and a co-cultivation temperature of 25-28℃.Under these optimized conditions,the number of transformants was significantly increased.The successful establishment of this high-efficiency ATMT system not only fills a critical gap in the genetic manipulation toolkit for the symbiotic fungus Ph506 but also provides a vital technical platform for subsequent investigations into gene function identification,secondary metabolite biosynthetic pathways,and the complex metabolic regulatory networks of this unique fungal strain.