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

【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C...

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

【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.