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| Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre/loxP system |
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YIN Yue-yan1, 2, HUA Meng-ying3, ZHAO Kuang-jie3, WAN Qiong-lian1, BU Shan4, LU Yu-wen3, ZHENG Hong-ying3, RAO Shao-fei3, YAN Fei3, PENG Jie-jun3, CHEN Hai-ru1, CHEN Jian-ping1, 3
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1 College of Plant Protection, Yunnan Agricultural University, Kunming 650201, P.R.China
2 Institute of Alpine Economic Plants, Yunnan Academy of Agricultural Sciences, Lijiang 674100, P.R.China
3 State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agroproducts, Institute of Plant Virology, Ningbo University, Ningbo 315211, P.R.China
4 Longping Branch of Graduate College, Hunan University, Changsha 410125, P.R.China
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摘要
本研究利用Cre/loxP重组系统构建了辣椒轻型斑驳病毒 (Pepper mild mottle virus, PMMoV)的嵌合体病毒载体。然而，在验证本氏烟中Cre重组酶瞬时表达量的研究中发现Cre重组酶可诱导本氏烟叶片坏死。为了缓解Cre重组酶引起的坏死表型，对本氏烟中瞬时表达Cre重组酶的表达系统进行了优化，构建了Cre表达后自敲除的瞬时表达载体 (ploxP-Cre)，有效缓解了Cre重组酶在本氏烟叶片上引起的坏死，可用于介导Cre/loxP重组系统侵染性克隆的重组。为了构建Cre/loxP重组系统的PMMoV侵染性克隆，将PMMoV基因组分段分别构建到独立的载体上获得了包含病毒基因组的载体pJM23 (1-5628 nt)，pJG1024 (5629-6356 nt)通过手足口病毒 (foot-and-mouth disease virus, FMDV)2A蛋白在PMMoV外壳蛋白(coat protein, CP)氮端 (N)融合了一个绿色荧光蛋白(modified green fluorescent protein, mGFP)，并分别在病毒基因组的重组区段插入2个同向的loxP位点和一半的内含子序列，在农杆菌介导下将pJM23、pJG1024和ploxP-Cre共转染本氏烟，转染8天后病毒发生了系统侵染，在系统侵染叶片中存在大小为300 nm×18 nm的杆状病毒粒子，实现了PMMoV病毒基因组在植物体内重组为功能性的病毒载体。进一步证实了Cre/loxP重组系统的PMMoV侵染性克隆与全长侵染性克隆的在转染4天后均可系统侵染，其系统叶病毒量无差别。同时该系统可用于研究不同病毒蛋白功能，通过构建番茄环纹斑点病毒 (Tomato zonate spot virus, TZSV)核壳体蛋白 (nucleocapsid, N) 基因的嵌合体病毒载体 (pJGTZSV)，在本氏烟中表达了N蛋白，并实现了系统侵染。我们的研究提供了一个新的方法用于嵌合体病毒和较大基因组的病毒侵染性克隆的构建。
Abstract
Cre/loxP, a site-specific recombination system, has been widely used for various purposes, including chromosomal translocations, generation of marker-free transgenic plants, tissue-specific activation of a reporter gene and efficient heterologous gene expression in plants. However, stable or transient expression of Cre recombinase in plants can cause chlorosis or necrosis. Here, we describe a modified Cre/loxP recombination system using a DNA fragment flanked with loxP sites in the same orientation in which necrosis induced by Cre recombinase in Nicotiana benthamiana leaves was alleviated. The modified system was successfully used to create functional GFP-tagged pepper mild mottle virus (PMMoV) and a chimeric virus with coat protein (CP) substitution assembled from separate pro-vector modules. Our results provide a new strategy and flexible technique to construct chimeric virus and infectious clones for plant viruses with large genomes.
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Received: 11 August 2021
Accepted: 05 November 2021
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| Fund: This work was supported by the National Key R&D Program of China (2019YFD1001800) and the China Agriculture Research System of MOF and MARA (CARS-24-C-04). This work was also supported by the Science & Technology Public Welfare Project of Ningbo City, China (202002N3005) and K. C. Wong Education Foundation, China. |
| About author: Correspondence PENG Jie-jun, E-mail: pengjiejun@yeah.net; CHEN Hai-ru, E-mail: 13908854041@163.com; CHEN Jian-ping, E-mail: jianpingchen@nbu.edu.cn |
Cite this article:
YIN Yue-yan, HUA Meng-ying, ZHAO Kuang-jie, WAN Qiong-lian, BU Shan, LU Yu-wen, ZHENG Hong-ying, RAO Shao-fei, YAN Fei, PENG Jie-jun, CHEN Hai-ru, CHEN Jian-ping.
2022.
Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre/loxP system. Journal of Integrative Agriculture, 21(8): 2456-2463.
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