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

第一作者： Feng, Chenwei

作者： Feng, Chenwei;Guo, Xiao;Gu, Tianxiao;Hua, Yanhong;Zhuang, Xinjian;Zhang, Kun;Zhuang, Xinjian;Zhang, Kun;Zhang, Kun;Zhang, Kun

作者机构：

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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