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| Rapid Construction of Infectious Clone of Pepper mottle virus Hangzhou Isolate Using an Escherichia coli-Agrobacterium tumefaciens-Saccharomyces cerevisiae Shuttle Vector |
| ZHAO Wei1,2, ZHANG Can2, FU Kang2, MA Ning2, GE Jia-Cheng3, SUN Kai2,*, YU Xiao-Ping2,* |
1 College of Metrology Measurement and Instrument, China Jiliang University, Hangzhou 310018, China;
2 Key Laboratory State Administration for Market Regulation (Microbiological Metrology, Measurement & Bio-product Quality Security), College of Life Sciences, China Jiliang University, Hangzhou 310018, China;
3 Hailir Pesticides and Chemicals Group Co., Ltd., Qingdao 266109, China |
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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.
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Received: 16 July 2024
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Corresponding Authors:
* sunkai0719@126.com; yxp@cjlu.edu.cn
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