利用Cre/loxP位点特异性重组系统构建PMMoV嵌合体病毒载体

doi:10.1016/S2095-3119(21)63864-9

Journal of Integrative Agriculture ›› 2022, Vol. 21 ›› Issue (8): 2456-2463.DOI: 10.1016/S2095-3119(21)63864-9

利用Cre/loxP位点特异性重组系统构建PMMoV嵌合体病毒载体

收稿日期:2021-08-11 接受日期:2021-11-05 出版日期:2022-08-01 发布日期:2021-11-05

Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre/loxP system

YIN Yue-yan^{1, 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-ping^{1, 3}

¹College 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
³State 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

Received:2021-08-11 Accepted:2021-11-05 Online:2022-08-01 Published:2021-11-05
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
Supported by:
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.

摘要/Abstract

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.

Key words: pepper mild mottle virus , Cre/loxP , necrosis , infectious cDNA clone , chimeric virus

. 利用Cre/loxP位点特异性重组系统构建PMMoV嵌合体病毒载体[J]. Journal of Integrative Agriculture, 2022, 21(8): 2456-2463.

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. Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre/loxP system[J]. Journal of Integrative Agriculture, 2022, 21(8): 2456-2463.

参考文献

Alonso E I, Garcia-Luque A, de la Cruz B, Wicke M J, Avila-Rincon M T, Serra C, Castresana C, Diaz-Ruiz J R. 1991. Nucleotide sequence of the genomic RNA of pepper mild mottle virus, a resistance-breaking tobamovirus in pepper. Journal of General Virology, 72, 2875–2884.
An Y Q, McDwell J M, Huang S R, McKinney E C, Chambliss S, Meagher R B. 1996. Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. The Plant Journal, 10,107–121.
Arumugam N, Gupta V, Jagannath A, Mukhopadhyay A, Pradhan A K, Burma P K, Pental D. 2007. A passage through in vitro culture leads to efficient production of marker-free transgenic plants in Brassica juncea using the Cre-loxP system. Transgenic research, 16, 703–712.
Avila-Rincon M J, Ferrero M L, Alonso E, Garcia I, Diaz-Ruiz J R. 1989. Nucleotide sequences of 5´ and 3´ non-coding regions of pepper mild mottle virus strain S RNA. Journal of General Virology, 70, 3025–3031.
Bendahmane M, Szecs J, Chen I, Berg R H, Beachy R N. 2002. Characterization of mutant tobacco mosaic virus coat protein that interferes with virus cell-to-cell movement. Proceedings of the National Academy of Sciences of the United States of America, 99, 3645–3650.
Berendsen S M H, chravesande W E W. 2020. Complete genome sequence of a novel genotype of Pepper mild mottle virus infecting pepper in chile. Microbiology Resource Announcements, 9, e01183-20.
Bordat A, Houvenaghel M C, German-Retana S. 2015. Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg. Virology Journal, 12, 89–97.
Cao B, Huang Z, Chen G, Lei J. 2010. Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system. Genetics and Molecular Biology, 33, 298–307.
Carpenter M A, Law E K, Serebrenik A, Brown W L, Harris R S. 2019. A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination. Methods, 156, 79–84.
Chen B Y, Lin Q, Yin Y Y, Jiang L L, Wang F, Hu S Z, Wu X Y, Xu F, Peng J J, Zheng H Y, Yan C Q, Mo X H, Chen J P, Yan F, Lu Y W. 2021. Construction of an infectious full-length cDNA clone of potato aucuba mosaic virus. Archives of Virology, 166, 1427–1431.
Coppoolse E R, de Vroomen M J, Roelofs D, Smit J, van Gennip F, Hersmus B J M, Nijkamp H J J, van Haaren M J J. 2003. Cre recombinase expression can result in phenotypic aberrations in plants. Plant Molecular Biology, 51, 263–279.
Desbiez C, Chandeysson C, Lecoq H, Moury B. 2012. A simple, rapid and efficient way to obtain infectious clones of potyviruses. Journal of Virology Methods, 183, 94–97.
Escalante C, Alcala-Briseno R I, Valverde R A. 2018. First report of a mixed infection of Pepper mild mottle virus and Tobacco mild green mosaic virus in pepper (Capsicum annuum) in the United States. Plant Disease, 102, 1469–1469.
Garcia-Luque I, Ferrero M L, Rodriquez J M, Alonso E, Cruz A D L, Sanz A I C, Vaquero C, Serra M T, Diaz-Ruiz J R. 1993. The nucleotide sequence of the coat protein genes and 3´ non-coding regions of two resistance-breaking tobamoviruses in pepper shows that they are different viruses. Archives of Virology, 131, 75–88.
Genda Y, Sato K, Nunomura O, Hirabayashi T, Tsuda S. 2011. Immunolocalization of Pepper mild mottle virus in developing seeds and seedlings of Capsicum annuum. Journal of General Plant Pathology, 77, 201–208.
Guo Y, Liu B C, Ding Z Z, Li G B, Liu M Z, Zhu D T, Sun Y N, Dong S S, Lou Z Y. 2017. Distinct mechanism for the formation of the ribonucleoprotein complex of tomato spotted wilt virus. Journal of Virology, 91, e00892-17.
Han K L, Zheng H Y, Ji M F, Cui W J, Hu S Z, Peng J J, Lin L, Liu Y, Chen J P, Yan F. 2020. A single amino acid in coat protein of Pepper mild mottle virus determines its subcellular localization and the chlorosis symptom on leaves of pepper. Journal of General Virology, 101, 565–570.
Hasiow-Jaroszewska B, Budzynska D, Zarzynska-Nowak A. 2019. First report of pepper mild mottle virus in peppers in Poland. Plant Disease, 103, 1441–1441.
Hron T, Spanielova H, Suchanova J, Forstova J. 2013. The Cre/loxP recombination system for production of infectious mouse polyomavirus. Virus Research, 176, 128–136.
Ikegashira Y, Ohki T, Ichiki U T, Higashi T, Hagiwara K, Omura T, Honda Y, Tsuda S. 2004. An immunological system for the detection of pepper mild mottle virus in soil from green pepper fields. Plant Disease, 88, 650–656.
Kasai Y, Harayama S. 2016. Construction of marker-free transgenic strains of chlamydomonas reinhardtii using a Cre/loxP-mediated recombinase system. PLoS ONE, 11, e0161733.
Li X D, An M N, Wu Y H. 2016. First report of Pepper mild mottle virus in Northeast China. Plant Disease, 100, 541–541.
Liang M T, Yang C P, Xie Z P, Staehelin C. 2012. Use of the Cre-loxP recombination system as an estimate for agrobacterium-mediated co-transformation of tobacco leaves. Biotechnology Letters, 34, 747–754.
Loonstra A, Vooijs M, Beverloo H B, Allak B A, van Drunen E, Kanaar R, Berns A, Jonkers J. 2001. Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America, 98, 9209–9214.
Lutz K A, Svab Z, Maliga P. 2006. Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system. Nature Protocols, 1, 900–910.
Lv Y, Edwards H J, Zhou J, Xu P. 2019. Combining 26s rDNA and the Cre-loxP system for iterative gene integration and efficient marker curation in yarrowia lipolytica. American Chemical Society Synthetic Biology, 8, 568–576.
Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y. 2004. In planta engineering of viral RNA replicons: Efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proceedings of the National Academy of Sciences of the United States of America, 101, 6852–6857.
Nishihama R, Ishida S, Urawa H, Kamei Y, Kohchi T. 2016. Conditional gene expression/deletion systems for marchantia polymorpha using its own heat-shock promoter and Cre/loxP-mediated site-specific recombination. Plant Cell Physiology, 57, 271–280.
Odell J T, Hoopes J L, Vermerris W. 1994. Seed-specific gene activation mediated by the Cre/lox site-specific recombination system. Plant Physiology, 106, 447–458.
Orbegozo J, Solorzano D, Cuellar W J, Bartolini I, Roman M L, Ghislain M, Kreuze J. 2016. Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi. Transgenic Research, 25, 813–828.
Ow D W, Medberry S L. 1995. Genome manipulation through site-specific recombination. Critical Reviews in Plant Sciences, 14, 239–261.
Peng J J, Shi B B, Zheng H Y, Lu Y W, Lin L, Jiang T, Chen J P, Yan F. 2015. Detection of pepper mild mottle virus in pepper sauce in China. Archives of Virology, 160, 2079–2082.
Que Q D, Wang H Y, Jorgensen R A. 1998. Distinct patterns of pigment suppression are produced by allelic sense and antisense chalcone synthase transgenes in petunia flowers. Plant Journal, 13, 401–409.
Sternberg N, Sauer B, Hoess R, Abremski K. 1986. Bacteriophage P1 Cre gene and its regulatory region: Evidence for multiple promoters and for regulation by DNA methylation. Journal of Molecular Biology, 187, 197–212.
Tsuda S, Kirita M, Watanabe Y. 1998. Characterization of a pepper mild mottle tobamovirus strain capable of overcoming the L3 gene-mediated resistance, distinct from the resistance-breaking Italian isolate. Molecular Plant–Microbe Interactions, 11, 327–331.
Velasco L, Janssen D, Ruiz-Garcia L, Segundo E, Cuadrado I M. 2002. The complete nucleotide sequence and development of a differential detection assay for a pepper mild mottle virus (PMMoV) isolate that overcomes L3 resistance in pepper. Journal of Virology Methods, 106, 135–140.
Wachsman G, Heidstra R. 2010. The CRE/lox system as a tool for developmental studies at the cell and tissue level. Molecular Biology Reports, 655, 47–64.
Yang C, Ge J, Fu X, Luo K, Xu C. 2021. Dual reproductive cell-specific promoter-mediated split-Cre/loxP system suitable for exogenous gene deletion in hybrid progeny of transgenic Arabidopsis. International Journal of Molecular Sciences, 22, 5080–5096.
Youssef F, Marais A, Faure C, Gentit P, Candresse T. 2011. Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study. Virology Journal, 8, 488.
Zhang Y, Liu H, Li B, Zhang J T, Li Y, Zhang H. 2009. Generation of selectable marker-free transgenic tomato resistant to drought, cold and oxidative stress using the Cre/loxP DNA excision system. Transgenic Research, 18, 607–619.
Zhao K J, Yin Y Y, Hua M Y, Wang S X, Mo X H, Yuan E P, Zheng H Y, Hou L X, Chen H R, Chen J P, Peng J J, Yan F. 2021. Pod pepper vein yellows virus, a new recombinant polerovirus infecting Capsicum frutescens in Yunnan province, China. Virology Journal, 18, 42.
Zheng H Y, Xiao C L, Han K L, Peng J J, Lin L, Lu Y W, Xie L, Wu X H, Xu P, Li G J, Chen J P, Yan F. 2015. Development of an agroinoculation system for full-length and GFP-tagged cDNA clones of cucumber green mottle mosaic virus. Archives of Virology, 160, 2867–2872.

利用Cre/loxP位点特异性重组系统构建PMMoV嵌合体病毒载体

Construction of chimeric viruses based on pepper mild mottle virus using a modified Cre/loxP system

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics