Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (20): 4476-4488.doi: 10.3864/j.issn.0578-1752.2026.20.004

• PLANT PROTECTION • Previous Articles     Next Articles

Complete Genome Sequencing and Infectious cDNA Clone Construction of Paprika Mild Mottle Virus Isolated from Shandong Province

HAN ZhiLei(), HONG Hao(), YIN Xiao, FANG Le, XU Shuai, LI XiangDong, JIANG ShanShan()   

  1. Institute of Plant Protection, Shandong Academy of Agricultural Sciences/Shandong Key Laboratory for Green Prevention and Control of Agricultural Pests, Ji’nan 250100
  • Received:2026-04-22 Accepted:2026-05-19 Online:2026-10-21 Published:2026-10-21
  • Contact: JIANG ShanShan

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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

Table 1

Primers used in this study"

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
21F CAACAACAAT TTTACAACAACAAC 58 6476
6496R GGGATTCGAACCCCTTTTTAC
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
CP-F ATGCCTTATA CTGTATCTTC TCC 56 11122
MP-R GATTAATTAAACGAATCAGATGAAAT
HAP-F2 TTCGTTTAATTAATCATGAAGACTAATCTTTTTCTC 56 1179
HAP-R2 TACAGTATAAGGCATGATTAAAACGTACTCGATGACG
UBC-F TTTCGGTCCTGATGATACTCCC 56 204
UBC-R CACAGAGCAAAGACTGGATTGA
β-TUB-F[17] GAGGGTGAGTGAGCAGTTC 56 167
β-TUB-R[17] CTTCATCGTCATCTGCTGTC

Fig. 1

Agarose gel electrophoretogram of PaMMV segmented cloning"

Fig. 2

Complete genome organization of PaMMV-CN"

Table 2

Nucleotide sequences identity analysis of PaMMV-CN with other PaMMV isolates and partial isolates of the genus Tobamovirus"

病毒名称
Virus name		 GenBank登录号GenBank
accession number		 来源
Source		 寄主
Host		 一致性Identity (%)
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

Fig. 3

Construction of phylogenetic tree based on the complete genome sequences of PaMMV-CN and representative species of the genus Tobamovirus"

Fig. 4

pCB301-PaMMV recombinant vector map and infectivity validation in N. benthamiana"

Fig. 5

Infectivity validation of pCB301-PaMMV in four types of pepper"

Fig. 6

pCB301-PaMMV-GFP recombinant vector map and expression validation in N. benthamiana"

Fig. 7

Expression validation of pCB301-PaMMV-GFP in ‘Qie Men’ sweet pepper"

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