基于基因组序列分析的烟草轻绿花叶病毒江苏辣椒分离物侵染性克隆构建

doi:10.3864/j.issn.0578-1752.2023.08.006

摘要/Abstract

摘要：

【目的】烟草花叶病毒属（Tobamovirus）是危害辣椒、烟草等茄科作物的主要病毒之一，严重影响蔬菜作物的种植和生产。本研究旨在探明侵染江苏省南京市辣椒的烟草轻绿花叶病毒（tobacco mild green mosaic virus，TMGMV）分离物（TMGMV-JS）的基因组结构特征、系统进化关系及其致病性，为TMGMV的防控提供科学依据。【方法】从江苏省南京市采集的辣椒病样，提取总RNA，利用TMGMV特异检测引物确认阳性后，设计病毒特异性全长引物扩增TMGMV-JS全基因序列，通过同源重组的方法克隆至pCB301植物表达载体上，获得TMGMV-JS分离物基因组序列和全长cDNA侵染性克隆。BLAST分析TMGMV-JS分离物与已报道分离物的同源性，利用MEGA7软件的邻接法进行系统进化分析。将侵染性克隆通过农杆菌浸润本氏烟和辣椒，经RT-PCR和Western blot检测验证侵染效果，测定TMGMV-JS分离物的致病性。【结果】侵染江苏省南京市辣椒的TMGMV全长序列为6 356 nt，编码4个功能蛋白，分别为126K复制相关蛋白、183K复制酶、运动蛋白MP和外壳蛋白CP。同源性分析结果显示TMGMV-JS与重庆分离物TMGMV-TN29（MF139550）同源性最高，厦门分离物（JX534224）次之，系统进化分析结果也显示TMGMV-JS与其他TMGMV聚于同一大分支，其中与重庆、厦门两个分离物在同一小分支，相对近缘。构建的pCB301-TMGMV-JS侵染性克隆载体可以系统侵染本氏烟，引起叶片黄化，植株系统性坏死；也可以系统侵染辣椒，引起叶片斑驳、卷曲和植株矮化等症状。【结论】侵染江苏省南京市辣椒的TMGMV-JS分离物基因组全长6 356 nt，与国内重庆、厦门TMGMV分离物具有较近的亲缘关系，同属于一个分支；构建的TMGMV-JS侵染性克隆可以系统侵染本氏烟和辣椒，在本氏烟上会导致系统性坏死。

关键词: 烟草轻绿花叶病毒, 分子特征, 侵染性克隆, 致病性分析

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

高晓晓, 涂丽琴, 杨柳, 刘亚楠, 高丹娜, 孙枫, 李硕, 章松柏, 季英华. 基于基因组序列分析的烟草轻绿花叶病毒江苏辣椒分离物侵染性克隆构建[J]. 中国农业科学, 2023, 56(8): 1494-1502.

GAO XiaoXiao, TU LiQin, YANG Liu, LIU YaNan, GAO DanNa, SUN Feng, LI Shuo, ZHANG SongBai, JI YingHua. Construction of an Infectious Clone of Tobacco Mild Green Mosaic Virus Isolate Infecting Pepper from Jiangsu Based on Genomic Clone[J]. Scientia Agricultura Sinica, 2023, 56(8): 1494-1502.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.08.006

https://www.chinaagrisci.com/CN/Y2023/V56/I8/1494

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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	524
TMGMV-ssF	TCATTTCATTTGGAGAGGCCTGTATGTTTTAATAGTTTTCGACAACAACA	55.0	6356
TMGMV-ssR	GAGATGCCATGCCGACCCGGGTGGGCCGCTACCCGCGGT	66.0	6356
pB-F	AGGCTTTACACTTTATGCTTC	48.5	216
pB-R	AAGGCGATTAAGTTGGGT	45.8	216

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 (%)
登录号 Accession number	分离物 Isolate	寄主 Host	国家/地区 Country/region	缩写 Abbr.	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