基于柑橘叶斑驳病毒的VIGE体系构建与应用

doi:10.3864/j.issn.0578-1752.2026.20.005

摘要/Abstract

摘要：

【目的】病毒介导的基因编辑（virus-induced genome editing，VIGE）技术在植物中已得到广泛应用，但在柑橘上的研究仍处于起步阶段。本研究旨在建立基于柑橘叶斑驳病毒（citrus leaf blotch virus，CLBV）的VIGE体系，以期为柑橘提供一种方便快速的基因编辑工具。【方法】基于病毒表达载体pCLBV202构建靶向烟草PDS的系列含或不含tRNA元件的gRNA递送载体，农杆菌介导注射接种本氏烟（Nicotiana benthamiana）转Cas9株系KQ334，通过表型观察、靶位点扩增及限制性内切酶酶切（PCR/RE）、Hi-TOM高通量测序分析编辑事件，明确tRNA元件的数量及位置对编辑效率的影响；针对四川胜红蓟曲叶病毒（ageratum leaf curl Sichuan virus，ALCScV）基因组不同功能区设计特异性sgRNA并利用所建VIGE体系进行递送，接种ALCScV侵染性克隆并通过qPCR检测病毒积累量，评估其抗病毒效果；以转Cas9锦橙为材料，通过发根农杆菌（Agrobacterium rhizogenes）K599介导的真空浸润法导入靶向柑橘PDS的VIGE载体，筛选阳性毛状根并进行基因编辑验证。【结果】pCLBV202-tRNA-NbPDS sgRNA-tRNA处理后4周引起了明显的白化表型，PCR/RE检测到特异性目标片段，测序显示靶标位点处出现了多种类型的碱基突变，包括单碱基插入、单或多碱基缺失，表明本氏烟KQ334发生了基因编辑事件；Hi-TOM高通量测序结果表明，pCLBV202-tRNA-NbPDS sgRNA诱导的突变率为13.54%—20.84%，显著优于不含tRNA元件的处理组（1.10%—2.94%）。ALCScV防控试验中，所有处理组均检测到病毒靶基因编辑事件；qPCR检测结果显示，各处理组的病毒积累量较对照组均有不同程度的下降，其中pCLBV202-tRNA-AL sgRNA1-tRNA在处理后3、5、7 d的病毒积累量较对照组均显著下降(P≤0.05)。利用转Cas9锦橙，通过发根农杆菌介导的真空浸润转化法获得pCLBV202-tRNA-CsPDS sgRNA阳性植株，PCR/RE检测结果初步验证已发生基因编辑。【结论】建立了基于柑橘叶斑驳病毒的VIGE体系，实现对烟草的高效基因编辑，并在柑橘中得到初步验证。该系统不依赖组织培养，具有应用于DNA病毒防控的潜力，并有望用于柑橘新种质创制。

关键词: 柑橘叶斑驳病毒, 病毒介导的基因编辑, CRISPR/Cas9

Abstract:

【Objective】Virus-induced genome editing (VIGE) has been widely applied in plants. However, its application in citrus remains at an early stage. This study aimed to establish a VIGE system based on citrus leaf blotch virus (CLBV), and to provide a convenient and rapid genome editing tool for citrus.【Method】A series of gRNA delivery vectors targeting the tobacco PDS, with or without tRNA elements, were constructed based on the viral expression vector pCLBV202. These recombinant constructs were delivered into Cas9-expressing Nicotiana benthamiana line KQ334 via Agrobacterium-mediated infiltration. Genome editing events were evaluated by phenotypic observation, target-site amplification followed by restriction enzyme digestion (PCR/RE), and Hi-TOM high-throughput sequencing to determine the effects of the number and position of tRNA elements on editing efficiency. For antiviral assessment, sgRNAs targeting different functional regions of the ageratum leaf curl Sichuan virus (ALCScV) genome were designed and delivered using the established VIGE system. Plants were inoculated with infectious ALCScV clones, and viral accumulation was determined by qPCR to evaluate antiviral efficacy. In addition, a CLBV-based VIGE vector targeting the citrus PDS was introduced into Cas9-transgenic ‘Jincheng’ sweet orange using Agrobacterium rhizogenes K599-mediated vacuum infiltration. Positive hairy roots were identified and subjected to molecular validation of genome editing.【Result】Following treatment with pCLBV202-tRNA-NbPDS sgRNA-tRNA, a pronounced albino phenotype was observed at 4 weeks post-inoculation. PCR/RE analysis detected the specific target fragment, and sequencing revealed multiple types of mutations at the target site, including single-base insertions and single- or multiple-base deletions, indicating that genome editing had occurred in N. benthamiana KQ334. Hi-TOM high-throughput sequencing further showed that the mutation frequency induced by pCLBV202-tRNA-NbPDS sgRNA ranged from 13.54% to 20.84%, significantly higher than that of the treatment group without tRNA elements (1.10%-2.94%). In the ALCScV antiviral assay, target gene editing events were detected in all treatment groups. qPCR analysis showed that viral accumulation decreased to varying degrees compared with the control, with viral accumulation in plants treated with pCLBV202- tRNA-AL sgRNA1-tRNA being significant reduction at 3, 5, and 7 days post-treatment (P≤0.05). Using Cas9-transgenic ‘Jincheng’ sweet orange, a positive pCLBV202-tRNA-CsPDS sgRNA plant was obtained via A. rhizogenes-mediated vacuum infiltration, and PCR/RE analysis preliminarily confirmed the occurrence of genome editing.【Conclusion】A CLBV-based VIGE system was established to achieve efficient gene editing of tobacco, which was preliminarily verified in citrus. The system is independent of tissue culture, shows potential for control of DNA viruses, and may provide a promising tool for the development of new citrus germplasm.

Key words: citrus leaf blotch virus (CLBV), virus-induced genome editing (VIGE), CRISPR/Cas9

王晓莲, 王春庆, 李佳欣, 李娅毓, 王丽婷, 丁子锐, 陈琪, 田旭斌, 宋震. 基于柑橘叶斑驳病毒的VIGE体系构建与应用[J]. 中国农业科学, 2026, 59(20): 4489-4499.

WANG XiaoLian, WANG ChunQing, LI JiaXin, LI YaYu, WANG LiTing, DING ZiRui, CHEN Qi, TIAN XuBin, SONG Zhen. Construction and Application of a Citrus Leaf Blotch Virus-Based Virus-Induced Genome Editing System[J]. Scientia Agricultura Sinica, 2026, 59(20): 4489-4499.

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参考文献 30

[1]	Haft D H, Selengut J, Mongodin E F, Nelson K E. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes[J]. PLoS Computational Biology, 2005, 1(6): e60. doi: 10.1371/journal.pcbi.0010060 pmid: 16292354
[2]	Demirci Y, Zhang B H, Unver T. CRISPR/Cas9: An RNA-guided highly precise synthetic tool for plant genome editing[J]. Journal of Cellular Physiology, 2018, 233(3): 1844-1859. doi: 10.1002/jcp.25970 pmid: 28430356
[3]	Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121): 819-823. doi: 10.1126/science.1231143 pmid: 23287718
[4]	Ran F A, Hsu P D, Wright J, Agarwala V, Scott D A, Zhang F. Genome engineering using the CRISPR-Cas9 system[J]. Nature Protocols, 2013, 8(11): 2281-2308. doi: 10.1038/nprot.2013.143 pmid: 24157548
[5]	Wang Q, Zhang D, Dai Y R, Liu C C. Efficient tobacco rattle virus- induced gene editing in tomato mediated by the CRISPR/Cas9 system[J]. Biotechnology Journal, 2024, 19(5): e2400204.
[6]	Mikhaylova E. Virus-induced genome editing (VIGE): One step away from an agricultural revolution[J]. International Journal of Molecular Sciences, 2025, 26(10): 4599. doi: 10.3390/ijms26104599
[7]	Oh Y, Kim H, Kim S G. Virus-induced plant genome editing[J]. Current Opinion in Plant Biology, 2021, 60: 101992. doi: 10.1016/j.pbi.2020.101992
[8]	Gentzel I N, Ohlson E W, Redinbaugh M G, Wang G L. VIGE: Virus-induced genome editing for improving abiotic and biotic stress traits in plants[J]. Stress Biology, 2022, 2(1): 2. doi: 10.1007/s44154-021-00026-x
[9]	Wang M, Gao S, Zeng W, Yang Y, Ma J, Wang Y. Plant virology delivers diverse toolsets for biotechnology[J]. Viruses, 2020, 12(11): 1338. doi: 10.3390/v12111338
[10]	Zhang C, Liu S, Li X, Zhang R, Li J. Virus-induced gene editing and its applications in plants[J]. International Journal of Molecular Sciences, 2022, 23(18): 10202. doi: 10.3390/ijms231810202
[11]	Jiang N, Zhang C, Liu J Y, Guo Z H, Zhang Z Y, Han C G, Wang Y. Development of beet necrotic yellow vein virus-based vectors for multiple-gene expression and guide RNA delivery in plant genome editing[J]. Plant Biotechnology Journal, 2019, 17(7): 1302-1315. doi: 10.1111/pbi.13055 pmid: 30565826
[12]	Qiao J H, Zang Y, Gao Q, Liu S N, Zhang X W, Hu W Y, Wang Y, Han C G, Li D W, Wang X B. Transgene- and tissue culture-free heritable genome editing using RNA virus-based delivery in wheat[J]. Nature Plants, 2025, 11: 1252-1259. doi: 10.1038/s41477-025-02023-8
[13]	Ma X, Zhang X, Liu H, Li Z. Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9[J]. Nature Plants, 2020, 6: 773-779. doi: 10.1038/s41477-020-0704-5 pmid: 32601419
[14]	Ali Z, Abulfaraj A, Idris A, Ali S, Tashkandi M, Mahfouz M M. CRISPR/Cas9-mediated viral interference in plants[J]. Genome Biology, 2015, 16: 238. doi: 10.1186/s13059-015-0799-6 pmid: 26556628
[15]	Vives M C, Martín S, Ambrós S, Renovell A, Navarro L, Pina J A, Moreno P, Guerri J. Development of a full-genome cDNA clone of citrus leaf blotch virus and infection of citrus plants[J]. Molecular Plant Pathology, 2008, 9(6): 787-797. doi: 10.1111/j.1364-3703.2008.00501.x pmid: 19019007
[16]	Liu H, Song S, Wu W, Mi W L, Shen C, Bai B X, Wu Y F. Distribution and molecular characterization of citrus leaf blotch virus from Actinidia in Shaanxi Province, China[J]. European Journal of Plant Pathology, 2019, 154(3): 855-862. doi: 10.1007/s10658-019-01677-3
[17]	Galipienso L, Navarro L, Ballester-Olmos J F, Pina J A, Moreno P, Guerri J. Host range and symptomatology of a graft-transmissible pathogen causing bud union crease of citrus on trifoliate rootstocks[J]. Plant Pathology, 2000, 49(2): 308-314. doi: 10.1046/j.1365-3059.2000.00449.x
[18]	崔甜甜, 晏建红, 宾羽, 李中安, 周常勇, 宋震. 利用酵母同源重组系统快速构建柑橘叶斑驳病毒的侵染性克隆[J]. 中国农业科学, 2018, 51(9): 1695-1705. DOI: 10.3864/j.issn.0578-1752.2018.09.007.
	Cui T T, Yan J H, Bin Y, Li Z A, Zhou C Y, Song Z. Construction of citrus leaf blotch virus infectious cDNA clone by yeast homologous recombination system[J]. Scientia Agricultura Sinica, 2018, 51(9): 1695-1705. DOI: 10.3864/j.issn.0578-1752.2018.09.007. (in Chinese)
[19]	张琦, 段玉, 苏越, 蒋琪琪, 王春庆, 宾羽, 宋震. 基于柑橘叶斑驳病毒的表达载体构建及应用[J]. 中国农业科学, 2022, 55(22): 4398-4407. DOI: 10.3864/j.issn.0578-1752.2022.22.006.
	Zhang Q, Duan Y, Su Y, Jiang Q Q, Wang C Q, Bin Y, Song Z. Construction and application of expression vector based on citrus leaf blotch virus[J]. Scientia Agricultura Sinica, 2022, 55(22): 4398-4407. DOI: 10.3864/j.issn.0578-1752.2022.22.006. (in Chinese)
[20]	段玉. 柑橘黄龙病RPA检测体系及柑橘叶斑驳病毒表达载体构建[D]. 重庆: 西南大学, 2021.
	Duan Y. Establishment of RPA detection system for HLB and construction of expression vector based on citrus leaf blotch virus[D]. Chongqing: Southwest University, 2021. (in Chinese)
[21]	Wang C, Ma Z, Zhou J, Li J, Cao P, Song C, Li C, Wang X, Bin Y, Zhou C, Song Z. A simple and effective VIGS system facilitates the control of citrus canker by silencing CsLOB1[J]. Phytopathology Research, 2024, 6: 12. doi: 10.1186/s42483-024-00234-z
[22]	Li P, Jing C, Ren H, Jia Z, Ghanem H, Wu G, Li M, Qing L. Analysis of pathogenicity and virulence factors of ageratum leaf curl Sichuan virus[J]. Frontiers in Plant Science, 2020, 11: 527787. doi: 10.3389/fpls.2020.527787
[23]	Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi J J, Qiu J L, Gao C. Targeted genome modification of crop plants using a CRISPR-Cas system[J]. Nature Biotechnology, 2013, 31(8): 686-688. doi: 10.1038/nbt.2650 pmid: 23929338
[24]	Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(11): 3570-3575. doi: 10.1073/pnas.1420294112 pmid: 25733849
[25]	Komura S, Kishi-Kaboshi M, Abe F, Inoue Y, Yoshida K. Improvement of simultaneous genome editing of homoeologous loci in polyploid wheat using CRISPR/Cas9 applying tRNA processing system[J]. Plant Biotechnology, 2025, 42(2): 167-172. doi: 10.5511/plantbiotechnology.25.0214b pmid: 40636432
[26]	Nekrasov V, Staskawicz B, Weigel D, Jones J D G, Kamoun S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease[J]. Nature Biotechnology, 2013, 31(8): 691-693. doi: 10.1038/nbt.2655
[27]	Veluthambi K, Sunitha S. Targets and mechanisms of geminivirus silencing suppressor protein AC2[J]. Frontiers in Microbiology, 2021, 12: 645419. doi: 10.3389/fmicb.2021.645419
[28]	Salazar-González J A, Castro-Medina M, Bernardino-Rivera L E, Martínez-Terrazas E, Casson S A, Urrea-López R. In-planta transient transformation of avocado (Persea americana) by vacuum agroinfiltration of aerial plant parts[J]. Plant Cell, Tissue and Organ Culture, 2023, 152(3): 635-646. doi: 10.1007/s11240-022-02436-9
[29]	刘亭君, 陈祥凤, 王俊利, 林敏, 董明右. 微型CRISPR核酸酶Cas12f研究进展及在基因编辑领域中的应用[J]. 中国人兽共患病学报, 2023, 39(2): 172-179. doi: 10.3969/j.issn.1002-2694.2022.00.185
	Liu T J, Chen X F, Wang J L, Lin M, Dong M Y. Research progress of miniature CRISPR nuclease Cas12f and its application in the field of gene editing[J]. Chinese Journal of Zoonoses, 2023, 39(2): 172-179. (in Chinese)
[30]	Harrington L B, Burstein D, Chen J S, Paez-Espino D, Ma E, Witte I P, Cofsky J C, Kyrpides N C, Banfield J F, Doudna J A. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes[J]. Science, 2018, 362(6416): 839-842. doi: 10.1126/science.aav4294 pmid: 30337455

引物名称Primer name	引物序列Primer sequence (5′-3′)	用途Usage
CLBV-JC-F	ATACCCAAGTCTAACTCAGTCAATA	CLBV检测引物 Primers for CLBV detection
CLBV-JC-R	CTCATAACTGCTTCCACGATT	CLBV检测引物 Primers for CLBV detection
PDS-Dete-F	AGAAATGTAGCCCGAAGGATTATGTGT	重组载体中sgRNA/tRNA插入片段检测引物 Primers for detecting sgRNA/tRNA inserts in the recombinant vector
tRNA-Dete-R	TTAAGAGTGTAAAGTCCTGGCC
NbPDS-TR12-F	GAAACACATCACCTAGGCGG	扩增烟草NbPDS靶标片段798 bp Amplification of the 798 bp target fragment of the tobacco NbPDS
NbPDS-TR12-R	GGGCGTGAGGAAGTACGAAA
ALCScV-Dete-F	ATGTGGGATCCATTGTTAAACGA	ALCScV检测引物 Primers for ALCScV detection
ALCScV-Dete-R	TCAGGGCTTTTGAACATCCTGTA	ALCScV检测引物 Primers for ALCScV detection
qPCR-F	CCCGTGTATGCTACTTTGAAAG	定量检测植株内ALCScV积累水平 Quantitative detection of ALCScV accumulation in plants
qPCR-R	ACCTCACACCTTCAGACTGG
CsPDS-3-P2-F	TGAACATTTCAATTACTTAGG	扩增柑橘CsPDS靶标片段482 bp Amplification of the 482 bp target fragment of citrus CsPDS
CsPDS-5-P1-R	AGGATTCTTCATTTTCAATGA