菌物学报2026，Vol.45Issue(8)：126-138,13.DOI:10.13346/j.mycosystema.260048
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
Establishment of an Agrobacterium tumefaciens-mediated transformation system for Penicillium herquei cultivated by attelabid weevils
摘要
Abstract
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.关键词
丝状真菌/遗传操作体系/植菌昆虫菌业/mCherry荧光蛋白Key words
filamentous fungi/genetic manipulation system/fungiculture by insects/mCherry fluorescent protein引用本文复制引用
晏露闻,邱鹏磊,刘晓静,魏东盛,刘杏忠..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[J].菌物学报,2026,45(8):126-138,13.基金项目
天津市科技重大专项与工程——全国重点实验室重大项目(24ZXZSSS00490) （24ZXZSSS00490）
南开大学引进人才科研启动经费(030/C029215002)This work was supported by the Tianjin Municipal Major Science and Technology Special Projects and Engineering-National Key Laboratory Major Projects(24ZXZSSS00490)and the Startup Fund from the Nankai University to XZL(030/C029215002). （030/C029215002）
