菌物学报2025，Vol.44Issue(8)：105-115,11.DOI:10.13346/j.mycosystema.250018
基于CRISPR/Cas9核蛋白复合体的芳香镰孢菌基因敲除体系
CRISPR/Cas9 ribonucleoprotein complex-based knockout system in Fusarium redolens
摘要
Abstract
Fusarium redolens,recognized as both a plant pathogenic and endophytic fungus,has been involved in numerous foundational biological studies;however,gene editing and genetic transformation systems of this fungus have not yet been reported.In this study,a genetic transformation system for F.redolens strain YP04 was established by utilizing the CRISPR/Cas9 ribonucleoprotein complex.An optimized protoplast preparation method was developed,and ribonucleoprotein(RNP)complexes targeting the c18.1_Beas and c18.1_Bea3 genes,which were implicated in beauvericin biosynthesis,were designed and prepared.Protoplasts were co-transformed with RNPs and donor DNA containing the hygromycin resistance gene(hyg),and in situ knockout mutants were successfully isolated through hygromycin resistance selection and PCR identification.Notably,the Δc18.1_Bea3 knockout mutants constituted 5.56%of resistance colonies when donor DNA was transformed exclusively,whereas the Δc18.1_Beas and Δc18.1_Bea3 mutants accounted for 22.22%and 25.00%,respectively,following co-transformation with RNPs.HPLC analysis revealed that the yields of beauvericin synthesized by the wild-type YP04 as well as Δc18.1_Beas and Δc18.1_Bea3 knockout mutants were 169.25,30.00,and 285.22 mg/L,respectively.The gene editing system established in this study holds significant potential for elucidating the functions of various pathogenic and secondary metabolic genes of F.redolens.关键词
芳香镰孢菌/CRISPR/Cas9核蛋白复合体/原生质体/基因敲除/白僵菌素Key words
Fusarium redolens/CRISPR/Cas9 ribonucleoprotein complex/protoplast/gene knockout/beauvericin引用本文复制引用
赵璟琛,商文静,曹燕霞,范三红,胡小平..基于CRISPR/Cas9核蛋白复合体的芳香镰孢菌基因敲除体系[J].菌物学报,2025,44(8):105-115,11.基金项目
科技部国际合作重点专项(KY202002018) （KY202002018）
国家现代农业(小麦)产业技术体系项目(CARS-03-37) （小麦）
留坝县西洋参研究专项(202300223302169)This work was supported by the Key International Cooperation Projects of the Ministry of Science and Technology(KY202002018),the National Modern Agricultural(Wheat)Industry Technology System(CARS-03-37),and the Liuba County's Special Research Project on American Ginseng(202300223302169). （202300223302169）
