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OA

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 Prov-ince,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 sys-tematic 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 pro-teins.Whole-genome nucleotide sequence alignment revealed that ToBRFV-MZ shared over 99%iden-tity 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 symp-toms 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 wild-type 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.