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

作　　者：赵薇 张灿 付康 马宁 葛家成 孙凯 俞晓平 ZHAOWei;ZHANG Can;FU Kang;MANing;GE Jia-Cheng;SUN Kai;YU Xiao-Ping(College of Metrology Measurement and Instrument,China Jiliang University,Hangzhou 310018,China;Key Laboratory State Administration for Market Regulation(Microbiological Metrology,Measurement&Bio-product Quality Security),College of Life Sciences,China Jiliang University,Hangzhou 310018,China;Hailir Pesticides and Chemicals Group Co.,Ltd.,Qingdao 266109,China)

机构地区：[1]中国计量大学计量测试与仪器学院,杭州310018 [2]中国计量大学生命科学学院国家市场监督管理总局重点实验室(微生物计量检测与生物制品质量安全),杭州310018 [3]海利尔药业集团股份有限公司,青岛266109

出　　处：《农业生物技术学报》2025年第2期443-452,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金面上项目(32472649);浙江省属高校基本科研业务费(2022YW04)。

摘　　要：辣椒斑驳病毒(Pepper mottle virus,PepMoV)是马铃薯Y病毒属(Potyvirus)的成员,能够侵染辣椒(Capsicum annuum)、番茄(Solanum lycopersicum)等重要茄科作物。本研究利用小RNA测序技术,在浙江省杭州市的辣椒样品中成功检测到PepMoV。通过RT-PCR技术扩增其基因组,获得长度为9640个核苷酸的全长基因组序列。为了克服PepMoV侵染性克隆构建过程中面临的大片段拼接困难及基因组含有大肠杆菌(Escherichia coli)致死基因等问题,采用酵母(Saccharomyces cerevisiae)体内同源重组技术,将5个DNA片段在酵母中一步组装成PepMoV病毒的全长侵染性克隆。重要的是,酵母质粒可以直接转化入农杆菌(Agrobacterium tumefaciens)并进行浸润接种,从而避免了大肠杆菌克隆步骤及其相关的毒性问题。结果表明,与传统的酶切连接方法相比,该构建策略将所需时间从数月缩短至仅1周,获得的PepMoVHZ病毒侵染性克隆能够有效感染本氏烟草(Nicotiana benthamiana),并且发病症状明显。PepMoV病毒侵染性克隆的成功构建不仅为建立该病毒的快速检测方法提供了阳性样品对照,而且为深入研究该病毒的基因功能及其与寄主的相互作用机制提供了重要的实验材料和手段。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,9640 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 PepMoVHZ 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 

分 类 号：S41-30[农业科学—植物保护]