Generation of a Triple-Shuttling Vector and the Application in Plant Plus-Strand RNA Virus Infectious cDNA Clone Construction
文献类型： 外文期刊
作者： Feng, Chenwei 1 ; Guo, Xiao 1 ; Gu, Tianxiao 1 ; Hua, Yanhong 1 ; Zhuang, Xinjian 1 ; Zhang, Kun 1 ;
作者机构： 1.Yangzhou Univ, Coll Plant Protect, Dept Plant Pathol, Yangzhou 225009, Peoples R China
2.Yangzhou Univ, Joint Int Res Lab Agr Agriprod Safety, Minist Educ China, Yangzhou 225009, Peoples R China
3.Guangdong Acad Agr Sci, Plant Protect Res Inst, Guangdong Prov Key Lab High Technol Plant Protect, Guangzhou 510640, Peoples R China
4.Nanjing Normal Univ, Coll Life Sci, Jiangsu Engn & Technol Res Ctr Microbiol, Jiangsu Key Lab Microbes & Funct Genom, Nanjing 210023, Peoples R China
关键词： triple-shuttling vector; plant virus infectious clone; homologous recombinant (HR)-based cloning; yeast
期刊名称：INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES （ 影响因子：5.6； 五年影响因子：6.2 ）
ISSN：
年卷期： 2023 年 24 卷 6 期
页码：
收录情况： SCI
摘要： Infectious cloning of plant viruses is a powerful tool for studying the reverse genetic manipulation of viral genes in virus-host plant interactions, contributing to a deeper understanding of the life history and pathogenesis of viruses. Yet, most of the infectious clones of RNA virus constructed in E. coli are unstable and toxic. Therefore, we modified the binary vector pCass4-Rz and constructed the ternary shuttle vector pCA4Y. The pCA4Y vector has a higher copy number in the E. coli than the conventional pCB301 vector, can obtain a high concentration of plasmid, and is economical and practical, so it is suitable for the construction of plant virus infectious clones in basic laboratories. The constructed vector can be directly extracted from yeast and transformed into Agrobacterium tumefaciens to avoid toxicity in E. coli. Taking advantage of the pCA4Y vector, we established a detailed large and multiple DNA HR-based cloning method in yeast using endogenous recombinase. We successfully constructed the Agrobacterium-based infectious cDNA clone of ReMV. This study provides a new choice for the construction of infectious viral clones.
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