甜椒轻斑驳病毒山东分离物的全序列测定及侵染性克隆构建

doi:10.3864/j.issn.0578-1752.2026.20.004

摘要/Abstract

摘要：

【目的】甜椒轻斑驳病毒（paprika mild mottle virus，PaMMV）属于烟草花叶病毒属（Tobamovirus），近年来在多个国家的辣椒上相继检出，但目前尚无国内分离物全基因组序列报道。本研究旨在探究PaMMV国内分离物的序列详情、遗传进化，并构建病毒基因组cDNA全长侵染性克隆，为病毒研究和防治提供基础材料和技术支撑。【方法】使用RT-PCR和RACE相结合的方法扩增PaMMV的全基因组序列，使用DNAMAN 8.0、SDTv 1.2和MEGA 6.0软件对序列进行一致性分析和遗传进化分析；利用同源重组方法进行基因组全长cDNA克隆构建，并构建基于侵染性克隆改造的GFP蛋白表达载体；利用农杆菌介导法和摩擦接种法验证PaMMV基因组全长cDNA克隆的系统侵染性。【结果】拼接获得一株PaMMV山东分离物（PaMMV-CN）的基因组全长序列（GenBank登录号：OR365081），全长6 524 nt，基因组由5′末端非编码区（untranslated region，UTR），4个分别编码复制相关蛋白（Rep）、RNA依赖的RNA聚合酶（RdRp）、运动蛋白（MP）、外壳蛋白（CP）的开放阅读框（open reading frame，ORF）以及3′末端非编码区（3′ UTR）组成；序列分析显示，PaMMV-CN与已报道的4个PaMMV分离物核苷酸序列一致性较高，为98.3%—99.6%；系统发育分析显示，PaMMV-CN和其他4个PaMMV分离物形成明显聚类，与同属的多种辣椒常发病毒聚在群体较大的group III；构建的PaMMV全长cDNA克隆（pCB301-PaMMV）可以系统侵染本氏烟，在本氏烟系统叶引起卷叶症状，系统叶提取的病毒粒子在电镜下可以观察到明显的杆状粒子；pCB301-PaMMV能侵染‘茄门’甜椒、‘天宇’朝天椒、‘8819’长线椒和‘好农11’泡椒，在‘茄门’甜椒上症状最为明显，表现斑驳症状和叶面坏死性斑点，在‘天宇’朝天椒、‘8819’长线椒和‘好农11’泡椒上引起不同程度的皱缩、花叶或疱斑症状；构建的携带GFP报告基因的表达载体（pCB301-PaMMV-GFP）能高效、系统侵染本氏烟和‘茄门’甜椒，在本氏烟上引起卷叶症状且在紫外下能观察到明显的绿色荧光，在‘茄门’甜椒上能引起斑驳症状，系统叶在紫外下能观察到绿色荧光。【结论】成功获得了一条PaMMV山东分离物PaMMV-CN全基因组序列，进而构建了侵染性克隆pCB301-PaMMV，能够系统、高效侵染本氏烟和辣椒，且能用于稳定表达外源蛋白GFP。明确了山东省PaMMV的序列详情，可为该病毒后续研究提供有力工具。

关键词: 甜椒轻斑驳病毒, 全基因组, 序列分析, 侵染性克隆, 绿色荧光蛋白

Abstract:

【Objective】Paprika mild mottle virus (PaMMV) belongs to the genus Tobamovirus. It has been continuously detected in pepper crops in multiple countries worldwide in recent years, whereas no complete genome sequence of Chinese domestic isolates has been reported to date. This study aimed to explore the sequence details and genetic evolution of PaMMV domestic isolates, and to construct an infectious clone of the full-length cDNA of the virus genome, so as to provide basic materials and technical support for virus research and prevention.【Method】The complete genome of PaMMV was amplified by RT-PCR combined with RACE. DNAMAN 8.0, STDv 1.2 and MEGA 6.0 were used for sequence identity and phylogenetic analyses. A full-length genomic cDNA clone was constructed by homologous recombination, and a GFP protein expression vector modified from the infectious clone was further constructed. Agrobacterium-mediated infiltration and mechanical rub inoculation were performed to validate the systemic infectivity of the PaMMV full-length genomic cDNA clone.【Result】The complete genome sequence of a PaMMV Shandong isolate (PaMMV-CN, GenBank accession number: OR365081) with a total length of 6 524 nt was obtained. The genome comprises a 5′ untranslated region (5′ UTR), four open reading frames (ORFs) separately encoding replication-associated protein (Rep), RNA-dependent RNA polymerase (RdRp), movement protein (MP), and coat protein (CP), and a 3′ untranslated region (3′ UTR). Sequence analysis indicated that PaMMV-CN exhibited high nucleotide identity (98.3%-99.6%) with four previously documented PaMMV isolates. PaMMV-CN clustered tightly with the other four PaMMV isolates, and all these isolates were grouped into the large clade group III, together with a variety of common pepper-infecting viruses belonging to the genus Tobamovirus. The constructed PaMMV full-length cDNA clone (pCB301-PaMMV) could systemically infect Nicotiana benthamiana and induce typical leaf rolling symptoms on systemic leaves. Typical rod-shaped viral particles were clearly observed under an electron microscope in extracts prepared from systemically infected N. benthamiana leaves. The pCB301-PaMMV clone could infect ‘Qie Men’ sweet pepper, ‘Tian Yu’ pod pepper, ‘8819’ long line pepper, and ‘Hao nong 11’ pickled pepper. Among the tested varieties, ‘Qie Men’ sweet pepper displayed the most severe symptoms, including leaf mottling and foliar necrotic spots, while the other three pepper varieties showed varying degrees of leaf shrinkage, mosaic, or blistering symptoms. The constructed expression vector carrying the GFP reporter gene (pCB301-PaMMV-GFP) could efficiently and systemically infect N. benthamiana and ‘Qie Men’ sweet pepper. It caused leaf rolling symptoms and obvious green fluorescence under ultraviolet (UV) light in systemic leaves of N. benthamiana, and induced mottling symptoms in ‘Qie Men’ sweet pepper, with green fluorescence observed under UV light in systemic leaves.【Conclusion】The complete genome sequence of the PaMMV Shandong isolate PaMMV-CN was successfully obtained, and the infectious clone pCB301-PaMMV was further constructed. The clone can efficiently and systemically infect N. benthamiana and pepper, and can be used for stable expression of the exogenous GFP protein. The sequence details of PaMMV in Shandong Province were clarified, which could provide a powerful tool for the follow-up study of the virus.

Key words: paprika mild mottle virus (PaMMV), complete genome, sequence analysis, infectious clone, green fluorescent protein

韩志磊, 洪浩, 阴筱, 房乐, 许帅, 李向东, 姜珊珊. 甜椒轻斑驳病毒山东分离物的全序列测定及侵染性克隆构建[J]. 中国农业科学, 2026, 59(20): 4476-4488.

HAN ZhiLei, HONG Hao, YIN Xiao, FANG Le, XU Shuai, LI XiangDong, JIANG ShanShan. Complete Genome Sequencing and Infectious cDNA Clone Construction of Paprika Mild Mottle Virus Isolated from Shandong Province[J]. Scientia Agricultura Sinica, 2026, 59(20): 4476-4488.

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	退火温度 Annealing temperature (℃)	目的长度 Length (bp)
PaMMV-CP-F^[8]	GAGTTCATAGAGGCAGTACC	56	800
PaMMV-CP-R^[8]	CTTCGATTTAAGTGGAGGGAT	56	800
21F	CAACAACAAT TTTACAACAACAAC	58	6476
6496R	GGGATTCGAACCCCTTTTTAC	58	6476
3′ RACE-1^[16]	A(15)GCTTGAGCTCGAGTCCTCGTCACTCTGCTCACTGG	—	—
d (G)^[16]	GCCACGCGTCGACTAGTACG(15)	50	—
3′-R1^[16]	CCAGTGAGCAGAGTGACG	50	—
3′-R2^[16]	GAGGACTCGAGCTCAAGC	56	—
5′-R1	CATAAGTCAATGAACCATAGGG	50	423
5′-R2	ATAGGCCTGTGAAACTACCAATG	56	302
3′-F1	CACGGTTAGTATTAGGGCTTG	50	468
3′-F2	CTACCGCAGCTACTACATAAG	56	344
HAP-F1	TCATTTCATT TGGAGAGGCCTGTAAAATTTATACAATACAACAACAACAAT TTTACAACAA CAAC	56	6563
HAP-R1	GAGATGCCATGCCGACCCGGGTGGGCCCCATACCCGGGGTAAAGGGGGGATTCGAACCCCTTTTTAC	56	6563
CP-F	ATGCCTTATA CTGTATCTTC TCC	56	11122
MP-R	GATTAATTAAACGAATCAGATGAAAT	56	11122
HAP-F2	TTCGTTTAATTAATCATGAAGACTAATCTTTTTCTC	56	1179
HAP-R2	TACAGTATAAGGCATGATTAAAACGTACTCGATGACG	56	1179
UBC-F	TTTCGGTCCTGATGATACTCCC	56	204
UBC-R	CACAGAGCAAAGACTGGATTGA	56	204
β-TUB-F^[17]	GAGGGTGAGTGAGCAGTTC	56	167
β-TUB-R^[17]	CTTCATCGTCATCTGCTGTC	56	167

病毒名称 Virus name	GenBank登录号GenBank accession number	来源 Source	寄主 Host	一致性Identity (%)
病毒名称 Virus name	GenBank登录号GenBank accession number	来源 Source	寄主 Host	Genome	5′ UTR	Rep	RdRP	MP	CP	3′ UTR
甜椒轻斑驳病毒 Paprika mild mottle virus	OR365081	中国山东 Shandong, China	辣椒 C. annuum	—	—	—	—	—	—	—
	AB089381	日本高知 Kochi, Japan	辣椒 C. annuum	99.6	100.0	99.6	99.6	99.3	99.6	99.5
	KX187305	以色列约旦河谷 Jordan Valley, Israel	辣椒 C. annuum	98.3	100.0	98.5	98.4	97.5	97.9	99.5
	OK181768	希腊伯罗奔尼撒 Peloponnese, Greece	辣椒 C. annuum	98.4	98.5	98.3	98.4	97.9	98.5	98.9
	OR233190	阿塞拜疆（未知） Azerbaijan (unknown)	辣椒 C. annuum	98.3	98.5	98.2	98.3	97.6	98.7	98.9
欧布达辣椒病毒 Obuda pepper virus	OR233194	匈牙利（未知） Hungary (unknown)	辣椒 C. annuum	79.6	90.8	78.7	79.9	79.1	80.1	78.1
辣椒轻斑驳病毒 Pepper mild mottle virus	AY859497	中国北京 Beijing, China	—	64.6	63.1	62.4	64.7	65.3	66.6	55.1
烟草轻绿花叶病毒 Tobacco mild green mosaic virus	KM596785	中国湖南 Hunan, China	辣椒 C. annuum	63.4	63.1	61.8	63.4	64.8	66.4	56.7
烟草花叶病毒 Tobacco mosaic virus	MG516107	中国沈阳 Shenyang, China	烟草 N. tabacum	64.9	53.8	64.6	66.2	62.3	61.1	59.4
番茄花叶病毒 Tomato mosaic virus	MF002489	中国山东 Shandong, China	番茄 S. lycopersicum	64.9	52.3	64.3	66.0	61.1	64.3	58.8
番茄斑驳花叶病毒 Tomato mottle mosaic virus	MW373515	中国山东 Shandong, China	番茄 S. lycopersicum	64.7	53.8	64.0	65.8	60.1	64.5	59.4