农业生物技术学报2025，Vol.33Issue(2)：443-452,10.DOI:10.3969/j.issn.1674-7968.2025.02.017
利用大肠杆菌-农杆菌-酵母三元穿梭载体快速构建辣椒斑驳病毒杭州分离株侵染性克隆
Rapid Construction of Infectious Clone of Pepper mottle virus Hangzhou Isolate Using an Escherichia coli-Agrobacterium tumefaciens-Saccharomyces cerevisiae Shuttle Vector
摘要
Abstract
Pepper mottle virus(PepMoV)is a member of the genus Potyvirus,capable of infecting important Solanaceous crops such as pepper(Capsicum annuum)and tomato(Solanum lycopersicum).In this study,PepMoV was successfully detected in pepper samples from Hangzhou,Zhejiang province,using small RNA sequencing technology.The complete genome,9 640 nucleotides in length,was amplified using RT-PCR.To overcome difficulties in constructing infectious clones of PepMoV,such as large fragment assembly challenges and lethal genes for Escherichia coli,yeast(Saccharomyces cerevisiae)homologous recombination technology was employed,which enabled the assembly 5 DNA fragments into the full-length infectious clone of PepMoV in a single step in yeast.Importantly,the yeast plasmid can be directly transformed into(Agrobacterium tumefaciens)for infiltration,thereby avoiding the E.coli cloning step and its associated toxicity issues.Results indicated that,compared with traditional enzyme ligation methods,this construction strategy reduced the required time from several months to just one week.The obtained infectious clone of PepMoV-HZ effectively infected Nicotiana benthamiana,exhibiting clear symptoms.The successful construction of the PepMoV infectious clone not only provides a positive sample control for the rapid detection method of the virus but also offers crucial experimental tools for in-depth studies of the virus's gene functions and its interaction mechanisms with the host.关键词
辣椒斑驳病毒/酵母体内重组/基因组/侵染性克隆/反向遗传学Key words
Pepper mottle virus/Yeast homologous recombination/Genome/Infectious clone/Reverse genetics分类
农业科技引用本文复制引用
赵薇,张灿,付康,马宁,葛家成,孙凯,俞晓平..利用大肠杆菌-农杆菌-酵母三元穿梭载体快速构建辣椒斑驳病毒杭州分离株侵染性克隆[J].农业生物技术学报,2025,33(2):443-452,10.基金项目
国家自然科学基金面上项目(32472649) （32472649）
浙江省属高校基本科研业务费(2022YW04) （2022YW04）
