摘要
中国小麦花叶病毒(Chinese wheat mosaic virus,CWMV)引起的小麦土传花叶病在山东烟台、威海等地危害严重。本研究克隆了CWMV烟台分离物的衣壳蛋白(Coat protein,CP)及富含半胱氨酸蛋白(Cysteine-rich protein,CRP)基因,并将其连接到原核表达载体p EHISTEV,转化大肠杆菌Rosetta。经IPTG诱导,表达出分子量均为19 k D的CP和CRP。将二者从凝胶中切下,乳化后免疫新西兰大耳兔4次,获得了两种蛋白的多克隆抗体。ELISA检测表明,CWMV CP和CRP抗血清的效价分别为1∶4096和1∶2048。Western blot分析证明该抗血清只与感染CWMV的小麦有特异性反应,而与健康或感染小麦黄花叶病毒的小麦无反应。利用含T3启动子的引物通过RT-PCR扩增出CWMV RNA2全长片段,经T/A克隆连接到p MD18-T,获得质粒p MD18-T-CWMV-RNA2。该质粒经XbaⅠ线性化后,利用T3 RNA聚合酶进行体外转录,转录产物摩擦接种本氏烟,15℃培养3天后,利用Western blot可从接种叶片中检测到瞬时表达的CWMV CP蛋白。
Chinese wheat mosaic virus（ CWMV） is a furovirus transmitted by Polymyxa graminis and induces soil- borne wheat mosaic disease. It has caused severe damage to winter wheat production in Yantai and Weihai of Shandong Province. The coat protein（ CP） and cysteine- rich protein（ CRP） genes of CWMV Yantai isolate were amplified by RT- PCR and cloned to prokaryotic expression vector p EHISTEV. The resultant plasmids were transformed into the competent cells of E. coil Rosetta. After induced with IPTG,both plasmids expressed protein with molecular weight of 19 k D. The New Zealand rabbits were immunized four times with CP and CRP cut from SDS- PAGE respectively,and the polyclonal antibody of the two proteins were ob-tained. The titers of antisera against CWMV CP and CRP were 1 ∶ 4096 and 1 ∶ 2048,respectively. In the Western blot assay,the antisera showed specific positive reaction only with wheat plants infected by CWMV,but not with those infected by WYMV or healthy ones. The full- length RNA2 of CWMV was amplified via RT- PCR with primer containing T3 promoter,and was linked to p MD18- T to produce the plasmid of p MD18- T- CWMV- RNA2. This plasmid was linearized with Xba Ⅰ and used as template for in vitro transcription with T3 RNA polymerase. The in vitro transcription product was mechanically inoculated to Nicotiana benthamiana leaves,and a band specific for CWMV CP was detected from the inoculated N. benthamiana leaves after three days.
出处
《山东农业科学》
2015年第8期6-10,共5页
Shandong Agricultural Sciences
基金
国家公益性行业(农业)科研项目(201303021)
山东省现代农业产业技术体系(SDAIT-01-022-09)
