Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (8): 1494-1502.doi: 10.3864/j.issn.0578-1752.2023.08.006

• PLANT PROTECTION • Previous Articles     Next Articles

Construction of an Infectious Clone of Tobacco Mild Green Mosaic Virus Isolate Infecting Pepper from Jiangsu Based on Genomic Clone

GAO XiaoXiao1,2(), TU LiQin1, YANG Liu1, LIU YaNan1, GAO DanNa1, SUN Feng1, LI Shuo1, ZHANG SongBai2, JI YingHua1()   

  1. 1 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
    2 College of Agriculture, Yangtze University/Hubei Engineering Research Center for Pest Forewarning and Management, Jingzhou 434025, Hubei
  • Received:2023-01-16 Accepted:2023-02-07 Online:2023-04-16 Published:2023-04-23

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Abstract:

【Objective】Tobamovirus is one of the main viruses that infect Solanaceae crops such as pepper and tobacco, which seriously affects the cultivation and production of crops. The purpose of this study is to investigate the genomic structural characteristics, phylogenetic relationship and pathogenicity of tobacco mild green mosaic virus (TMGMV) isolate infected pepper in Nanjing City of Jiangsu Province (TMGMV-JS), and to provide a scientific basis for the prevention and control of TMGMV. 【Method】Total RNA was extracted from disease pepper samples, then the positive samples were confirmed by specific detection primers of TMGMV. Subsequently, a pair of primers were designed and used to amplify the full-length genome sequence of TMGMV-JS isolate. The infectious cDNA clone of TMGMV-JS isolate was obtained by cloning the amplified products into pCB301 vector by homologous recombination. Then the homology of TMGMV-JS isolate with reported isolates was analyzed by BLAST and the neighbor-joining method of MEGA7 software was used for the phylogenetic analysis. Nicotiana benthamiana and Capsicum annuum were infiltrated with the infectious cDNA clone mediated by Agrobacterium tumefaciens. Furthermore, RT-PCR and Western blot were determined to define the pathogenicity of TMGMV-JS isolate.【Result】TMGMV-JS isolate contains 6 356 nt, encoding four functional proteins, 126K replication-associated protein, 183K replicase, movement protein MP, and coat protein CP. Homology analysis showed that TMGMV-JS isolate shared the highest identity with Chongqing TMGMV-TN29 isolate (MF139550), followed by Xiamen isolate (JX534224). Phylogenetic analysis showed that TMGMV-JS was clustered in a big branch with other TMGMV isolates and relatively closed to Chongqing and Xiamen isolates in a small branch. Furthermore, the constructed pCB301-TMGMV-JS infectious cDNA clone can systematically infect N. benthamiana, causing leaf yellow and systemic necrosis symptoms. It can also systematically infect C. annuum, causing symptoms such as leaf mottle, curling and dwarfing symptoms. 【Conclusion】The full-length genome of TMGMV-JS isolate infecting C. annuum from Nanjing City, Jiangsu Province is 6 356 nt, which is closely related to Chongqing and Xiamen isolates and belongs to the same branch. Significantly, the constructed infectious clone of TMGMV-JS can systemically infect N. benthamiana and C. annuum, causing systemic necrosis symptoms on N. benthamiana.

Key words: tobacco mild green mosaic virus (TMGMV), molecular characteristic, infectious clone, pathogenicity analysis

Table 1

Primers used in this study"

引物名称
Primer name		 序列
Sequence (5′-3′)		 退火温度
Annealing temperature (℃)		 片段长度
Fragment length (bp)
524-F CGTCATCGAGTACGTTTTAA 49.4 524
524-R AGGAAATCTCACAACAATAG 45.6
TMGMV-ssF TCATTTCATTTGGAGAGGCCTGTATGTTTTAATAGTTTTCGACAACAACA 55.0 6356
TMGMV-ssR GAGATGCCATGCCGACCCGGGTGGGCCGCTACCCGCGGT 66.0
pB-F AGGCTTTACACTTTATGCTTC 48.5 216
pB-R AAGGCGATTAAGTTGGGT 45.8

Table 2

Nucleotide and amino acid sequence identities between TMGMV-JS and nine TMGMV isolates and other five viruses in Tobamovirus"

登录号
Accession number		 分离物
Isolate		 寄主
Host		 国家/地区
Country/region		 缩写
Abbr.		 同源性Identity (%)
基因组<BOLD>G</BOLD>enome 183K 126K MP CP
MF139550 TMGMV-TN29 辣椒C. annuum 中国重庆Chongqing, China TMGMV 99.83 99.88 99.91 100.00 100.00
JX534224 Xiamen 辣椒C. annuum 中国福建厦门
Xiamen, Fujian, China		 TMGMV 99.76 99.50 99.37 100.00 100.00
MT675965 DSMZ PV-0112 粉蓝烟草N. glauca 美国USA TMGMV 99.28 99.63 99.55 100.00 100.00
MN267903 WW17-E1-Nocc-pos 西方烟N. occidentalis 斯洛文尼亚Slovenia TMGMV 98.85 99.32 99.19 98.44 100.00
MZ405634 DSMZ PV-0124 普通烟N. tabacum 意大利Italy TMGMV 97.62 98.51 98.11 97.66 98.74
DQ821941 HP 辣椒C. annuum 中国台湾Taiwan, China TMGMV 97.11 97.95 97.39 96.48 99.37
AB078435 Japanese 辣椒C. annuum 日本Japan TMGMV 97.11 98.14 97.66 97.27 99.37
KM596785 HN 辣椒C. annuum 中国湖南长沙
Changsha, Hunan, China		 TMGMV 98.36 97.76 97.66 96.09 100.00
MK005155 BR 粉蓝烟草N. glauca 巴西Brazil TMGMV 97.48 98.26 97.84 97.27 99.37
NC_002692 Queensland 番茄Lycopersicon esculentum 澳大利亚Australia ToMV 64.70 68.78 65.80 57.42 70.44
NC_022230 MX5 番茄Solanum lycopersicum 墨西哥Mexico ToMMV 64.35 68.47 65.17 55.08 71.07
NC_001367 U1 普通烟N. tabacum 美国USA TMV 63.04 67.91 64.81 55.56 71.07
NC_003630 S 辣椒C. annuum 西班牙Spain PMMoV 62.04 66.96 62.97 62.11 70.06
NC_004422 Arabidopsis 油菜Brassica napus 西班牙Spain YoMV 58.94 65.85 62.01 38.65 43.31

Fig. 1

The phylogenetic tree based on the complete genomic sequence of TMGMV The genomes of TMGMV isolates and other classic strains of tobamoviruses were downloaded from NCBI. MEGA7 software was used to construct phylogenetic tree by neighbor-joining method. : TMGMV Jiangsu isolate sequenced in this study "

Fig. 2

Schematic diagram of TMGMV-JS cDNA infectious clone"

Fig. 3

Pathogenicity of pTMGMV-JS infectious clone on N. benthamiana"

Fig. 4

Pathogenicity of pTMGMV-JS infectious clone on C. annuum"

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