农业生物技术学报2025，Vol.33Issue(9)：2036-2048,13.DOI:10.3969/j.issn.1674-7968.2025.09.014
利用纳米磁珠介导的茄链格孢菌遗传转化技术的建立及其泛素化蛋白酶基因AsPR45功能研究
Development of a Magnetic Nanoparticle-mediated Genetic Transformation Technology for Alternaria solani and Functional Analysis of the Ubiquitin-proteasome Gene AsPR45
摘要
Abstract
The ubiquitin-proteasome system(UPS)is a critical protein degradation pathway in fungal cells,playing a vital role in regulating protein homeostasis and maintaining cellular functions.Previous studies have shown that during the early stages(0～72 h)of Alternaria solani infection in potato(Solanum tuberosum),the expression of the AsPR45 gene,which encoded the P45 subunit of the 26S proteasome in A.solani,was significantly upregulated.To further explore the function of the AsPR45 gene,this study first optimized a genetic transformation method for A.solani mediated by magnetic nanoparticles(MNPs).Using this transformation assay,a premature termination construct of AsPR45 was developed based on the pTOR-mRFP vector backbone and introduced into the wild-type strain TA-0410.Functional validation was carried out on 2 positive mutant strains.The transformation efficiency was the highest when the mass ratio of MNPs to the pTOR-mRFP vector was 1∶4,with binding at 37℃for 30 min,followed by co-incubation with spores at room temperature for 1.5 h,achieving a transformation rate of(36.77±2.91)%.Biological function assays revealed that premature termination mutants of AsPR45 exhibited no significant difference in hyphal growth rate compared with the wild-type strain.However,the mutants produced less melanin in hyphae,had a significantly reduced conidiation rate,and showed markedly decreased pathogenicity on the leaves of potato cultivar'Desiree'.Additionally,the EC50 values for difenoconazole resistance differed significantly between the wild-type strain and the mutants.These results indicated that AsPR45 was a key gene in the ubiquitination pathway in A.solani,playing an important role in fungal morphogenesis,potato infection,and stress resistance.This study not only provides a technical foundation for functional gene research in A.solani but also contributes to the identification of potential fungicide targets,accelerating the development of strategies for controlling potato early blight disease.关键词
茄链格孢/纳米磁珠/转化/泛素-蛋白酶体途径/突变体Key words
Alternaria solani/Nanoparticles/Transformation/Ubiquitin-proteasome pathway/Mutant分类
农业科技引用本文复制引用
赵玉娇,付爱华,张佳滢,刘文雯,杨巧梅,姜冬美,唐唯,李健美..利用纳米磁珠介导的茄链格孢菌遗传转化技术的建立及其泛素化蛋白酶基因AsPR45功能研究[J].农业生物技术学报,2025,33(9):2036-2048,13.基金项目
云南省基础研究重点项目(202301AS070010) （202301AS070010）
