柑橘鳞皮病毒基因组全长cDNA克隆构建及其侵染性鉴定

doi:10.3864/j.issn.0578-1752.2025.17.007

摘要/Abstract

摘要：

【目的】柑橘鳞皮病毒（citrus psorosis virus，CPsV）是蛇形病毒科（Aspiviridae）蛇形病毒属（Ophiovirus）的一种三分体负义单链RNA病毒，可引起柑橘树干开裂流胶，甚至整株死亡，严重威胁柑橘产业安全。负链RNA病毒的反向遗传学体系构建具有挑战性，本研究旨在建立CPsV基因组的全长cDNA克隆，并鉴定其侵染性，为其致病机理等研究打下基础。【方法】利用软件Primer 5设计引物，以CPsV侵染植株总核酸为模板，分别进行CPsV 3条链RNA1、RNA2、RNA3的RT-PCR扩增。基于双元表达载体pXT1，通过In-Fusion同源重组技术分别构建3条RNA链的cDNA克隆，进行酶切验证并测序分析。利用本氏烟（Nicotiana benthamiana）接种体系筛选CPsV 3条链的cDNA克隆组合，并进一步通过农杆菌介导注射接种至草本寄主千日红（Gomphrena globosa），真空浸润接种至不同的柑橘品种，观察其症状并进行分子检测。【结果】分别获得CPsV RNA1全长cDNA克隆2个，RNA2全长cDNA克隆2个，RNA3全长cDNA克隆2个。随机选取RNA1、RNA2、RNA3全长cDNA克隆各一个进行组合，作为CPsV基因组全长cDNA克隆，通过农杆菌介导注射接种本氏烟并进行RT-PCR检测。结果表明8个组合中CPsV-122阳性率最高，为62.50%。核苷酸序列分析显示，CPsV-122与西班牙分离物P-121的序列一致性最高，其RNA1、RNA2及RNA3相应的序列一致性分别为98.06%、97.10%和99.32%。在基于外壳蛋白氨基酸序列构建的系统进化树上，CPsV-122与P-121聚在同一支，并与来自中国、突尼斯、意大利的5个分离株聚为一簇。通过农杆菌介导接种CPsV-122至草本寄主千日红及柑橘品种尤力克柠檬（Citrus limon）和邓肯葡萄柚（C. paradise），进行症状观察和RT-PCR检测。结果表明，7 dpi时，千日红阳性率为16.67%（2/12），在25 dpi时，阳性植株出现明显的红褐色枯斑、叶片局部坏死等CPsV侵染症状；邓肯葡萄柚和尤力克柠檬的RT-PCR检测结果除阳性对照外均呈阴性，但在90 dpi，CPsV-122接种的20株尤力克柠檬植株中有13株表现出明显的矮化、黄化和嫩梢充胶、枯死等CPsV侵染典型症状，而空载对照组和健康对照组均未观察到特异性症状。【结论】CPsV-122为CPsV基因组全长cDNA克隆，能够系统侵染千日红并在柑橘上引起植株矮化和嫩梢枯死等CPsV侵染典型症状。

关键词: 柑橘鳞皮病毒, 全长cDNA克隆, 农杆菌介导接种, 侵染性鉴定, 柑橘病毒

Abstract:

【Objective】Citrus psorosis virus (CPsV) is a tripartite, negative-sense single-stranded RNA virus of the genus Ophiovirus in the family Aspiviridae, which can cause cracking of citrus trunks and even the death of the whole plant, seriously threatening the safety of the citrus industry. The construction of reverse genetics systems for negative-strand RNA viruses is challenging. This study aims to establish a full-length cDNA clone of CPsV genome and determine its infectivity, with the expectation of laying a foundation for research on its pathogenic mechanism and other aspects.【Method】The primers were designed using software Primer 5, and the total nucleic acid of CPsV-infected plants was used as a template for RT-PCR amplification of the three strands of CPsV, RNA1, RNA2 and RNA3, respectively. Based on the dual-expression vector pXT1, cDNA clones of three RNA strands were constructed by In-Fusion homologous recombination technology, which were verified by enzyme digestion and sequencing analysis. The cDNA clones of the three strands of CPsV were screened by the Nicotiana benthamiana inoculation system, and further inoculated into the herb host Gomphrena globosa by Agrobacterium-mediated injection, and then inoculated into different citrus varieties by vacuum infiltration to observe their symptoms and perform molecular detection.【Result】A total of two CPsV RNA1 full-length cDNA clones, two RNA2 full-length cDNA clones, and two RNA3 full-length cDNA clones were obtained, respectively. One full-length cDNA clone of RNA1, RNA2 and RNA3 was randomly selected and combined as a full-length cDNA clone of CPsV genome, eight RNA1, RNA2 and RNA3 cDNA clones were selected and inoculated on N. benthamiana through Agrobacterium-mediated inoculation and RT-PCR detection. Among the eight combinations, the positive rate of CPsV-122 was the highest (62.50%). Sequence analysis showed that CPsV-122 had the highest sequence identity with Spanish isolate P-121, and the corresponding sequence identities of RNA1, RNA2 and RNA3 were 98.06%, 97.10% and 99.32%, respectively. In a phylogenetic tree based on the amino acid sequence of coat protein, CPsV-122 and P-121 were clustered in the same clade and five isolates from China, Tunisia and Italy. CPsV-122 was inoculated by Agrobacterium-mediated inoculation to G. globosa, Citrus limon and C. paradise for symptom observation and RT-PCR test. The results showed that at 7 dpi, the positive rate of G. globosa was 16.67% (2/12), and at 25 dpi, the positive plants showed obvious reddish-brown blight, local necrosis of leaves and other CPsV infection symptoms. The RT-PCR results of C. paradise and C. limon were negative except for the positive control, but at 90 dpi, 13 out of 20 C. limon plants inoculated with CPsV-122 showed obvious CPsV infection symptoms such as dwarfing, yellowing, wilting shoots and shoot gelatinization, while no specific symptoms were observed in the empty vector group and the healthy control group.【Conclusion】CPsV-122 is a full-length cDNA clone of the CPsV genome, which can systematically infect G. globosa and cause typical CPsV infection symptoms on citrus, such as dwarfing and withering of shoots.

Key words: citrus psorosis virus (CPsV), full-length cDNA clone, Agrobacterium-mediated inoculation, infectivity identification, citrus virus

李娅毓, 王新亮, 周金环, 李楚欣, 李佳欣, 田旭斌, 宋震. 柑橘鳞皮病毒基因组全长cDNA克隆构建及其侵染性鉴定[J]. 中国农业科学, 2025, 58(17): 3451-3460.

LI YaYu, WANG XinLiang, ZHOU JinHuan, LI ChuXin, LI JiaXin, TIAN XuBin, SONG Zhen. Construction and Infectivity Identification of Genome-Length cDNA of Citrus Psorosis Virus[J]. Scientia Agricultura Sinica, 2025, 58(17): 3451-3460.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2025.17.007

https://www.chinaagrisci.com/CN/Y2025/V58/I17/3451

图/表 9

表1

图1

图2

图3

图4

图5

图6

图7

图8

参考文献 32

[1]	MARTÍN S, ALIOTO D, MILNE R G, GARNSEY S M, GARCÍA M L, GRAU O, GUERRI J, MORENO P. Detection of citrus psorosis virus by ELISA, molecular hybridization, RT-PCR and immunosorbent electron microscopy and its association with citrus psorosis disease. European Journal of Plant Pathology, 2004, 110(7): 747-757.
[2]	WALLACE J M. Virus-strain interference in relation to symptoms of psorosis disease of citrus. Hilgardia, 1957, 27: 223-246.
[3]	VELAZQUEZ K, PINA J A, NAVARRO L, MORENO P, GUERRI J. Association of citrus psorosis B symptoms with a sequence variant of the citrus psorosis virus RNA 2. Plant Pathology, 2012, 61(3): 448-456.
[4]	DE FRANCESCO A, COSTA N, GARCIA M L. Citrus psorosis virus coat protein-derived hairpin construct confers stable transgenic resistance in citrus against psorosis A and B syndromes. Transgenic Research, 2017, 26(2): 225-235.
[5]	ACHACHI A, AIT BARKA E, IBRIZ M. Recent advances in citrus psorosis virus. Virus Disease, 2014, 25(3): 261-276.
[6]	邱柱石, 黄邦良. 广西的柑桔病毒病和类似病毒病害的发生与防治. 广西园艺, 2008, 19(6): 29-32.
	QIU Z S, HUANG B L. The occurrence and control of citrus virus diseases and similar viral diseases in Guangxi. Guangxi Horticulture, 2008, 19(6): 29-32. (in Chinese)
[7]	冯明峰. 植物分节段负链RNA病毒反向遗传学体系的突破与创新研究[D]. 南京: 南京农业大学, 2020.
	FENG M F. Establishment of the reverse genetics system for two representative segmented plant negative-strand RNA viruses[D]. Nanjing: Nanjing Agricultural University, 2020. (in Chinese)
[8]	王雁翔. 侵染猕猴桃的负义RNA病毒的鉴定及编码蛋白的互作研究[D]. 武汉: 华中农业大学, 2021.
	WANG Y X. Identification of negative sense RNA viruses infecting kiwifruit and study on interactions of proteins of these virus[D]. Wuhan: Huazhong Agricultural University, 2021. (in Chinese)
[9]	LUNA G R, PEÑA E J, BORNIEGO M B, HEINLEIN M, GARCÍA M L. Citrus psorosis virus movement protein contains an aspartic protease required for autocleavage and the formation of tubule-like structures at plasmodesmata. Journal of Virology, 2018, 92(21): e00355-18.
[10]	VELAZQUEZ K, RENOVELL A, COMELLAS M, SERRA P, GARCIA M L, PINA J A, NAVARRO L, MORENO P, GUERRI J. Effect of temperature on RNA silencing of a negative-stranded RNA plant virus: Citrus psorosis virus. Plant Pathology, 2010, 59(5): 982-990.
[11]	BELABESS Z, SAGOUTI T, RHALLABI N, TAHIRI A, MASSART S, TAHZIMA R, LAHLALI R, JIJAKLI M H. Citrus psorosis virus: Current insights on a still poorly understood Ophiovirus. Microorganisms, 2020, 8: 1197.
[12]	LUNA G R, PEÑA E J, BORNIEGO M B, HEINLEIN M, GARCIA M L. Ophioviruses CPsV and MiLBVV movement protein is encoded in RNA 2 and interacts with the coat protein. Virology, 2013, 441(2): 152-161.
[13]	NAUM-ONGANÍA G, GAGO-ZACHERT S, PEÑA E, GRAU O, GARCIA M L. Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase. Virus Research, 2003, 96(1/2): 49-61.
[14]	MORENO P, GUERRI J, GARCÍA M L. The psorosis disease of citrus: A pale light at the end of the tunnel. Journal of Citrus Pathology, 2015, 2(1): 28860.
[15]	刘文涛, 江禹, 李泽鸿. 反向遗传学技术在负链RNA病毒中的应用. 黑龙江畜牧兽医, 2010(7): 45-47.
	LIU W T, JIANG Y, LI Z H. The application of reverse genetics technology in negative-sense RNA viruses. Heilongjiang Animal Science and Veterinary Medicine, 2010(7): 45-47. (in Chinese)
[16]	SALEM R, ARIF I A, SALAMA M, OSMAN G E H. Polyclonal antibodies against the recombinantly expressed coat protein of the citrus psorosis virus. Saudi Journal of Biological Sciences, 2018, 25(4): 733-738.
[17]	MARTÍN S, LÓPEZ C, GARCÍA M L, NAUM-ONGANÍA G, GRAU O, FLORES R, MORENO P, GUERRI J. The complete nucleotide sequence of a Spanish isolate of citrus psorosis virus: Comparative analysis with other ophioviruses. Archives of Virology, 2005, 150(1): 167-176.
[18]	李敏, 周天宇, 张松, 杨方云, 周彦, 周常勇, 李中安, 曹孟籍. 柑橘鳞皮病毒3个分离物全基因组序列分析. 园艺学报, 2018, 45(10): 2030-2036.
	LI M, ZHOU T Y, ZHANG S, YANG F Y, ZHOU Y, ZHOU C Y, LI Z A, CAO M J. Complete genome sequence analysis of three citrus psorosis virus isolates. Acta Horticulturae Sinica, 2018, 45(10): 2030-2036. (in Chinese)
[19]	许建建, 王艳娇, 段玉, 马志敏, 宾羽, 周常勇, 宋震. 柑橘脉突病毒基因组全长cDNA克隆及其侵染性鉴定. 中国农业科学, 2020, 53(18): 3707-3715. doi: 10.3864/j.issn.0578-1752.2020.18.007.
	XU J J, WANG Y J, DUAN Y, MA Z M, BIN Y, ZHOU C Y, SONG Z. Construction of genome-length cDNA of citrus vein enation virus and identification of its infectivity. Scientia Agricultura Sinica, 2020, 53(18): 3707-3715. doi: 10.3864/j.issn.0578-1752.2020.18.007. (in Chinese)
[20]	崔甜甜. 柑橘黄化脉明病毒和柑橘叶斑驳病毒的侵染性克隆构建[D]. 重庆: 西南大学, 2018.
	CUI T T. Construction of infectious cDNA clones of citrus yellow vein clearing virus and citrus leaf blotch virus[D]. Chongqing: Southwest University, 2018. (in Chinese)
[21]	蒋琪琪, 许建建, 苏越, 张琦, 曹鹏, 宋晨虎, 李中安, 宋震. 柑橘黄化花叶病毒侵染性克隆构建及应用. 中国农业科学, 2022, 55(24): 4840-4850. doi: 10.3864/j.issn.0578-1752.2022.24.005.
	JIANG Q Q, XU J J, SU Y, ZHANG Q, CAO P, SONG C H, LI Z A, SONG Z. Construction and application of infectious clone of citrus yellow mosaic virus. Scientia Agricultura Sinica, 2022, 55(24): 4840-4850. doi: 10.3864/j.issn.0578-1752.2022.24.005. (in Chinese)
[22]	王艳娇. 柑橘脉突病毒侵染性克隆构建及其基因沉默抑制子鉴定[D]. 重庆: 西南大学, 2017.
	WANG Y J. Construction of infectious cDNA clones and identification of gene silencing suppressor of citrus vein enation virus[D]. Chongqing: Southwest University, 2017. (in Chinese)
[23]	XIAO Y X, DUTT M, MA H J, XIAO C, TONG Z, WANG Z Q, HE X J, SUN Z H, QIU W M. Establishment of an efficient root mediated genetic transformation method for gene function verification in citrus. Scientia Horticulturae, 2023, 321: 112298.
[24]	GARNSEY S M, TIMMER L W. Mechanical transmissibility of citrus ringspot virus isolates from Florida, Texas, and California// International Organization of Citrus Virologists Conference Proceedings (1957-2010), 1980, 8(8): 174-179.
[25]	吕婵娟, 周常勇, 周彦, 唐科志. 运用一步法RT-PCR检测柑橘鳞皮病毒及其在橘橙不同部位中的全年分布. 植物保护, 2007, 33(3): 42-45.
	LÜ C J, ZHOU C Y, ZHOU Y, TANG K Z. Detection of citrus psorosis virus by one-step RT-PCR and its annual distribution in different parts of Dweet tangor. Plant Protection, 2007, 33(3): 42-45. (in Chinese)
[26]	SIMEONE M, GÓMEZ C, BERTALMÍO A, RUIZ E, HAUTEVILLE C, SUAREZ L G, TITO B, GARCÍA M L. Detection of citrus psorosis virus by RT-qPCR validated by diagnostic parameters. Plant Pathology, 2021, 70(4): 980-986.
[27]	JUNG M, KIM N H, OH S H, KIM K H. Development of TaqMan-based real-time qPCR method for accurate detection and quantification of citrus psorosis virus and cytoplasmic-type citrus leprosis virus in saplings. The Plant Pathology Journal, 2024, 40(6): 625-632.
[28]	苏越. 柑橘花叶病毒侵染性克隆构建及其诱导的基因沉默体系建立[D]. 重庆: 西南大学, 2023.
	SU Y. Construction of infectious clones for citrus mosaic virus and development of a virus-induced gene silencing system[D]. Chongqing: Southwest University, 2023. (in Chinese)
[29]	李敏, 周天宇, 吴佳星, 周彦, 曹孟籍, 李中安. 柑橘鳞皮病毒RT-LAMP检测方法的建立与应用. 园艺学报, 2019, 46(7): 1409-1416.
	LI M, ZHOU T Y, WU J X, ZHOU Y, CAO M J, LI Z A. Establishment and application of loop-mediated isothermal amplification on citrus psorosis virus. Acta Horticulturae Sinica, 2019, 46(7): 1409-1416. (in Chinese)
[30]	乔兴华, 陈力, 孙海均, 易天凤, 张昕, 杨祥. 柑桔鳞皮病研究进展. 中国南方果树, 2022, 51(5): 195-199.
	QIAO X H, CHEN L, SUN H J, YI T F, ZHANG X, YANG X. Research progress on citrus psorosis virus disease. South China Fruits, 2022, 51(5): 195-199. (in Chinese)
[31]	FANG X D, YAN T, GAO Q, CAO Q, GAO D M, XU W Y, ZHANG Z J, DING Z H, WANG X B. A cytorhabdovirus phosphoprotein forms mobile inclusions trafficked on the actin/ER network for viral RNA synthesis. Journal of Experimental Botany, 2019, 70(15): 4049-4062.
[32]	LIU Q, ZHAO C L, SUN K, DENG Y L, LI Z H. Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes. Molecular Plant, 2023, 16: 616-631.

引物名称Primer name	引物序列Primer sequence (5′-3′)	用途Usage
CPsV1(354)-F1	ctaaggaaggtgtcagaatgg	CPsV-RNA1的2054 nt片段扩增 2054 nt fragment amplification of CPsV-RNA1
CPsV1(2409)-R1	accaacaagaggaaatgcc
CPsV1(2251)-F2	gaataagaaatgcgcccaca	CPsV-RNA1的2894 nt片段扩增 2894 nt fragment amplification of CPsV-RNA1
CPsV1(5125)-R2	tcaaggaggcaaaagg
CPsV1(5040)-F3	ccatcatgtcatccaacatca	CPsV-RNA1的3078 nt片段扩增 3078 nt fragment amplification of CPsV-RNA1
CPsV1(8118)-R3	acttacacaaatggggattgg
CPV-RNA1(JC)-F1	aagaccagagggctattgaga	CPsV-RNA1检测 CPsV-RNA1 detection
CPV-RNA1(JC)-R1	ggatcttgaattgtcattgtgctg	CPsV-RNA1检测 CPsV-RNA1 detection
CPV-RNA2(JC)-F1	tatctgtcacttcatcgaaagg	CPsV-RNA2检测 CPsV-RNA2 detection
CPV-RNA2(JC)-R1	gtcagcagtatttccatccttt	CPsV-RNA2检测 CPsV-RNA2 detection
CPV-RNA3(JC)-F1	tccaaccacgtcttcaaagcaa	CPsV-RNA3检测 CPsV-RNA3 detection
CPV-RNA3(JC)-R1	tatcagttatcttaatgttgctgg	CPsV-RNA3检测 CPsV-RNA3 detection
CPsV-RNA1-24K-F	aagtaagggttgaaaACTTACACAAATGGGGATTGGA	CPsV-RNA1全长序列扩增 CPsV-RNA1 full-length sequence amplification
CPsV-RNA1-RdRp-R	atcttgaacccctaggaaacttctgAAGCCTTCAGACAATATG
CPsV-RNA2-F3	tcacgttgacaggttatcac	CPsV-RNA2全长序列扩增 CPsV-RNA2 full-length sequence amplification
CPsV-RNA2-R3	tcatcctggtgacatttccac
CPsV-RNA3-F2	agtctcacagctaatcgatgCAAAAGCAAAATGGGAAAGCG	CPsV-RNA3全长序列扩增 CPsV-RNA3 full-length sequence amplification
CPsV-RNA3-R2	catagtcgatgctaccccaAAGATACCTTTGAGTCCAT