Sequences and infectious clones of tomato aspermy virus strain isolated from beijing | 番茄不孕病毒BJ株系基因组测定与侵染性克隆

Chinese. 番茄不孕病毒(Tomato aspermy virus, TAV)可侵染包括藜科(Chenopodiaceae)、茄科(Solanaceae)等在内的24个双子叶家族和3个单子叶家族的100多种植物，是具有重要经济价值的植物病毒之一。为研究TAV BJ株系(Tomato aspermy virus, TAV-BJ)的基因组功能，本实验对TAV-BJ基因组克隆测序，并构建侵染性克隆。以TAV-BJ侵染心叶烟(Nicotiana glutinosa)的总RNA为模板，RT-PCR获得其RNA2和RNA3；以TAV-BJ的dsRNA为模板，RT-PCR获得全长RNA1，目的片段PCR产物克隆测序获得TAV-BJ基因组全序列信息。RNA1全长3 409 nt，编码994个氨基酸的1a蛋白；RNA2 全长3 023 nt，含2个开放阅读框(open reading frame, ORF)，2a ORF编码829个氨基酸的2a蛋白，2b ORF编码78个氨基酸的2b蛋白；RNA3全长为2 216 nt，包含2个ORF，3a ORF 编码247个氨基酸的3a蛋白，外壳蛋白(coat protein, CP)ORF编码219个氨基酸的CP蛋白(TAV-BJ基因组RNA1、2和3 GenBank登录号分别为HQ424163，HQ424164和HQ424165)。TAV-BJ基因组cDNA克隆体外转录成RNA并接种于心叶烟上，结果表明转录产物在寄主上的症状反应和TAV-BJ病毒粒子RNA的接种相一致，TAV-BJ基因组cDNA侵染性克隆具有活性。由TAV-BJ各个基因片段与缺失2b基因的黄瓜花叶病毒Fny株系(Cucumber mosaic virus, CMV-FnyΔ2b)构建的假重组病毒接种于心叶烟，结果显示TAV-BJ的RNA2和RNA3能恢复CMV-FnyΔ2b在寄主上症状反应。嵌合型RNA3F3aTcp和RNA3T3aFcp的症状反应结果表明，F1F2Δ2bRNA3T3aFcp在寄主上产生花叶症状与F1F2Δ2bT3相一致。本研究获得TAV-BJ的基因组序列，成功构建侵染性克隆，同时发现TAV-BJ的3a基因具有CMV-Fny的2b基因的某些功能。

English. Tomato mosaic virus (TAV) is an important pathogen in agriculture, infecting more than 100 species of 24 dicotyledoneae and 3 monocotyledoneae, such as Chenopodiaceae and Solanaceae. In order to analyze the role of genomic RNAs, full length sequence of TAV isolated from Beijing (TAV-BJ) was obtained by RT-PCR and infectious cDNA clones was constructed. Using total RNA from Nicotiana glutinosa infected by TAV-BJ as template, full length of RNA2 and RNA3 were amplified by RT-PCR, and RNA1 was obtained from cDNA of TAV-BJ double stand RNA1. PCR products of RNA1, RNA2 and RNA3 were cloned into pUC118 vector and sequenced. TAV-BJ RNA1 contained 3 409 nucleotides (nts), encoding 1a protein of 994 amino acids, RNA2 was 3 032 nts, encoding 2a protein of 829 amino acids and 2b protein of 78 amino acids and RNA3 consisted of 2 218 nts and encoded 3a protein of 247 amino acids and coat protein (CP) of 219 amino acids (GenBank accession No of TAV-BJ genomic RNA1, 2 and 3 was HQ424163, HQ424164 and HQ424165, respectilvely). cDNA clones of TAV-BJ genomic RNAs were transcribed into RNA and the mixture of transcripts RNA was mechanically inoculated onto N. glutinosa. After some days of post-inoculation (dpi), T1T2T3 induced host plants expressed mosaic symptom, which was in accordance with that appearing on N. glutinosa infected by TAV-BJ viral RNAs. Seedlings of N. glutinosa infected by CMV-Fny lack of 2b gene (CMV-FnyΔ2b) were symptomless. Co-inoculation of T1, T2, T2Δ2b or T3 respectively with CMV-FnyΔ2b showed that the pseudorecombinant virus with T2 or T3 could resue the pathogenicity of CMV-FnyΔ2b in host plant, thus RNA3 of TAV-BJ might paly some function of 2b protein of CMV-FnyΔ2b. TAV-BJ RNA3 chimera infectious clones that replaced with 3a or CP gene fragment of CMV-Fny were constructed by Overlapping PCR. Biological assays of F1F2Δ2bRNA3T3aFcp and F1F2Δ2bRNA3 F3aTcp suggested that TAV 3a protein could compensate some function of 2b protein.In this study, infectious cDNA clones of TAV-BJ are successfully constructed and results of pseudorecombinant virus between CMV-Fny and TAV-BJ confirm that 3a gene of TAV-BJ have some role of CMV-Fny 2b gene.