ToMV番茄坏死株系农杆菌侵染性克隆构建及其表达  

Construction of Agrobacterium-mediated Infectious Clone and Its Expression of Tomatomosaic virus (ToMV) Strain

作　　者：刘妮娜[1] 程晓东[1] 卢冉[1] 廖乾生[1] 

机构地区：[1]浙江理工大学生命科学学院生物工程研究所,杭州310018

出　　处：《农业生物技术学报》2014年第8期1027-1034,共8页Journal of Agricultural Biotechnology

基　　金：浙江省自然基金项目(NO.Y3080228);浙江省生物学重中之重学科建设项目

摘　　要：摘要番茄花叶病毒（Tomato mosaic virus，ToMV）是烟草花叶病毒属（Tobamovirus）的典型成员，该病毒侵染严重影响农作物的产量和质量。ToMV-N5株系能引起番茄（Lycopersicon esculentum）主栽品种（合作903）系统性坏死，建立该株系的侵染性克隆体系是研究其致病机理的必要条件。本研究将TOMV-N5株系的基因组克隆至含35S启动子瞬时表达载体pCB301中，农杆菌（Agrobacterium tumefaciens）浸润接种本氏烟（Nicotiana benthamiana）和番茄结果表明，农杆菌侵染性克隆pCB301-ToMV-N5引起本氏烟和番茄系统性坏死症状，与其病毒粒子摩擦接种所引起的症状相一致。以绿色荧光蛋白基因（green fluorescent protein，GFP）代替ToMV-N5的外壳蛋白基因（coat protein，CP）获得表达载体pCB301-ToMV-N5CP45GFP，病毒接种结果表明，ToMV-N5能高效表达GFP，在病毒浸润接种浓度（OD600）为0．002时，GFP也能够有效表达。pCB301-TOMV-N5CP45GFP缺失了CP基因使得病毒不能系统性侵染，将烟草花叶病毒（Tobacco mosaic virus，TMV）U5株系包含CP大小为647nt（nt 5498～6145）片段克隆到pCB301-ToMV-N5CP45GFP中，获得pCB301-ToMV-N5CP45GFP-CP^U5，浸润接种本氏烟结果表明，病毒能长距离移动，在寄主非接种部位表达GFP；浸润及摩擦接种番茄结果表明，只能在显微镜下观察到微弱GFP，且局限于接种部位。本研究成功获得了ToMV-N5株系的农杆菌侵染性克隆及其表达载体，为研究该病毒侵染的分子机理提供了基础资料。Tomato mosaic virus （ToMV）, a type species of the genus Tobamovirus, severely affects the yield and quality of crops. ToMV-N5 infection caused Lycopersicon esculentum Mill. main cultivar Hezuo 903 systemic necrosis. It is essential to obtain its viral infectious clone for analyzing the mechanism of ToMV-N5 pathogenesis. In this study, the full length of ToMV-N5 genomic RNA was cloned into pCB301 vector which carried duplicated 35S promoter and ribozyme and then pCB301-ToMV-N5 was transformed into Agrobacterium tumefaciens GV3101. Agroinfiltration results indicated that the seedlings of Nicotiana benthemiana and L.esculentum infected by pCB301-ToMV-N5 appreared the same phenotypic caused by ToMV-N5 viral particles. Vector pCB301-ToMV-N5CP45GFP was constructed by replacing the ORF of viral coat protein gene （CP） with that of green fluorescent protein gene （GFP） and the data showed that the vectorcould highly express GFP in leaves of N. benthemiana in the presence of gene silencing suppressor p 19. Even in leaves infiltrated with A. tumefaciens cells diluted 1 ： 500 from a starting optical density （OD600） of 1.0, most cells of the infiltrated zone expressed GFP at 7 days post-infitation（dpi）. Due to the lack of CP, pCB301- ToMV-N5CP45GFP could not move to upper leaves of host plants, pCB301-ToMV-N5CP45GFP-CP^U5 was obtained by inserting CP （nt 5 498～6 145） of Tobacco mosaic virus （TMV） U5 strain and the phenotype of pCB301-ToMV-N5CP45GFP-CP^U5 on N. benthamiana suggested that the recombinant ToMV-N5 could move to non-inoculated leaves and GFP the whole plant expressed in the whole plant at 10 dpi. While the results from the seedlings of L.esculentum inoculated with pCB301-ToMV-N5 CP45GFP-CP^U5 suggested that there was only some weak GFP expression on inoculated leaves under the microscope. The successful construct of Agrobacterium-mediated infectious clone and its expression vector provides basic data for dissecting the molecular mechanism of ToMV-N5 replication and movement.

关 键 词：番茄花叶病毒(ToMV) 系统性坏死 农杆菌侵染性克隆 表达载体 

分 类 号：S432.41[农业科学—植物病理学]