农业生物技术学报2011，Vol.19Issue(6)：1120-1126,7.DOI:10.3969/j.issn.1674-7968.2011.06.019
番茄不孕病毒BJ株系基因组测定与侵染性克隆
Sequences and Infectious Clones of Tomato aspermy virus Strain Isolated from Beijing
摘要
Abstract
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 la protein of 994 amino acids, RNA2was 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, respectively). cDNA clones of TAV-BJ genomic RNAs were transcribed into RNA and the mixture of transcripts RNA was mechanically inoculated onto N. glutinosa. At days post-inoculation (dpi), T1T2T3 induced host plants to express mosaic symptom, which was in accordance with that appearing on N. gLuiinosa infected by TAV-BJ viral RNAs. Seedlings of N. gtutinosa infected by CMV-Fny lack of 2b gene (CMV-FnyA2b) was symptomless. Co-inoculation of Tl, T2, T2A2b or T3 respectively with CMV-FnyA2b showed that the pseudorecombinant virus with T2 or T3 could resue the pathogenicity of CMV-FnyA2b in host plant, thus RNA3 of TAV-BJ might paly some function of 2b protein of CMV-FnyA2b. 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 FlF2A2bRNA3T3aFcp and FlF2A2bRNA3 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.关键词
番茄不孕病毒/基因组序列/侵染性克隆/3a基因Key words
Tomato aspermy virus/ Sequence of genomic RNAs/ Infectious clones/ 3a gene引用本文复制引用
施伟,金圣塔,张海峰,王婷,陈集双,廖乾生..番茄不孕病毒BJ株系基因组测定与侵染性克隆[J].农业生物技术学报,2011,19(6):1120-1126,7.基金项目
本研究由浙江省自然科学项目(No.Y3080228)和浙江理工大学科研启动项目资助 （No.Y3080228）
