番茄褐色皱果病毒蒙自分离物的全基因组序列分析及侵染性克隆构建 Click here to download the full text Citation：杨洁,曹喜舟,杨桂香,杨薏卉,陈鹏,谭冠林,兰平秀,李凡.番茄褐色皱果病毒蒙自分离物的全基因组序列分析及侵染性克隆构建.Journal of Plant Protection,2026,53(4):955-968 Hits: Download times: Author Name Affiliation E-mail Yang Jie  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Cao Xizhou  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Yang Guixiang  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Yang Yihui  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Chen Peng  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Tan Guanlin  Modern Educational Technology Center, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Lan Pingxiu  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Li Fan  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China  fanli@ynau.edu.cn  中文摘要:为解析云南省蒙自市番茄褐色皱果病毒（tomato brown rugose fruit virus，ToBRFV）番茄分离物的基因组特征及系统进化关系，并建立其反向遗传学体系，通过反转录聚合酶链式反应（reverse transcription polymerase chain reaction，RT-PCR）扩增获得ToBRFV蒙自分离物ToBRFV-MZ的全基因组序列，分析其序列特征及系统发育关系，利用酵母Saccharomyces cerevisiae同源重组技术构建其全长cDNA克隆，并通过农杆菌Agrobacterium介导的浸润法在本氏烟、番茄和辣椒中验证其侵染性。结果表明：ToBRFV-MZ分离物基因组全长为6 396 nt（GenBank登录号为PV832075），编码4个功能蛋白。全基因组核苷酸序列一致性分析显示，该分离物与其他已报道ToBRFV分离物的一致性均高于99%，其中与中国Yinchuan分离物（GenBank登录号为OR500698）的一致性最高，为99.73%。系统发育分析显示，ToBRFV-MZ分离物与中国Yinchuan分离物和JS-2022分离物（Gen-Bank登录号为OR593752）的亲缘关系最近，聚为同一亚支。侵染性测试结果显示，所构建的pCB301-2μ-ToBRFV-MZ cDNA克隆能够系统侵染本氏烟，诱导叶片产生黄化、皱缩、向外卷曲和萎蔫等典型症状；电镜观察结果表明，该克隆产生的病毒粒体在形态和大小上均与野生型ToBRFV-MZ一致。该侵染性克隆在番茄上引起叶片卷曲、褪绿和畸形症状，在辣椒上引起系统叶坏死且植株矮缩，致病力与野生型ToBRFV-MZ相当，且系统叶中病毒RNA和外壳蛋白积累水平无显著差异。本研究获得了ToBRFV-MZ分离物的全基因组序列，构建的pCB301-2μ-ToBRFV-MZ全长cDNA克隆对寄主植物具有系统侵染能力。 中文关键词:番茄褐色皱果病毒  基因组结构  系统进化分析  侵染性克隆  致病性   Genome sequence analysis and infectious clone construction of the ToBRFV-MZ isolate Author Name Affiliation E-mail Yang Jie  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Cao Xizhou  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Yang Guixiang  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Yang Yihui  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Chen Peng  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Tan Guanlin  Modern Educational Technology Center, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Lan Pingxiu  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China    Li Fan  School of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan Province, China  fanli@ynau.edu.cn  Abstract:This study aimed to clarify the genomic features and phylogenetic relationships of a tomato brown rugose fruit virus (ToBRFV) isolate responsible for field outbreaks in Mengzi City, Yunnan Province, and to establish its reverse genetics system. The complete genome sequence of the ToBRFV-MZ isolate was obtained via reverse transcription-polymerase chain reaction (RT-PCR), followed by systematic analysis of its sequence characteristics and phylogenetic affinity. Furthermore, a full-length cDNA clone of ToBRFV-MZ was constructed through homologous recombination in Saccharomyces cerevisiae. Its infectivity was tested in Nicotiana benthamiana, Solanum lycopersicum, and Capsicum annuum with Agrobacterium-mediated infiltration. The results showed that the complete genome of ToBRFV-MZ was 6 396 nt in length (GenBank accession no. PV832075), encoding four functional proteins. Whole-genome nucleotide sequence alignment revealed that ToBRFV-MZ shared over 99% identity with other reported ToBRFV isolates, with the highest identity of 99.73% to the Yinchuan isolate from China (GenBank accession no. OR500698). Phylogenetic analysis further demonstrated that ToBRFV-MZ was most closely related to the Yinchuan and the JS-2022 isolate (GenBank accession no. OR593752), and these isolates clustered in the same subclade. Infectivity assays indicated that the pCB301-2μ -ToBRFV-MZ cDNA clone systemically infected N. benthamiana, inducing typical symptoms such as yellowing, leaf crinkling, outward curling, and wilting. Transmission electron microscopy showed that the virions produced by this clone were similar in morphology and size to those of wildtype ToBRFV-MZ. The ToBRFV-MZ infectious clone induced leaf curling, chlorosis, and deformation symptoms in tomato plants, and it caused leaf necrosis along with plant stunting in pepper plants. Its pathogenicity was comparable to that of the wild-type ToBRFV-MZ, with no significant differences in viral RNA and coat protein accumulation in systemically infected leaves. In conclusion, this study acquired the complete genome sequence of the ToBRFV-MZ isolate, and the constructed full-length cDNA clone pCB301-2μ-ToBRFV-MZ could establish systemic infection in host plants. keywords:tomato brown rugose fruit virus  genome structure  phylogenetic analysis  infectious clone  pathogenicity View Full Text  View/Add Comment  Download reader

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