Wrzesińska Barbara, Wieczorek Przemysław, Obrępalska-Stęplowska Aleksandra
Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection - National Research Institute, Władysława Węgorka 20 St, 60-318, Poznań, Poland.
Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection - National Research Institute, Władysława Węgorka 20 St, 60-318, Poznań, Poland.
J Virol Methods. 2016 Nov;237:179-186. doi: 10.1016/j.jviromet.2016.09.011. Epub 2016 Sep 19.
Full-length cDNA clones of Peanut stunt virus strain P (PSV-P) were constructed and introduced into Nicotiana benthamiana plants via Agrobacterium tumefaciens. The cDNA fragments corresponding to three PSV genomic RNAs and satellite RNA were cloned into pGreen binary vector between Cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator employing seamless recombinational cloning system. The plasmids were delivered into A. tumefaciens, followed by infiltration of hosts plants. The typical symptoms on systemic leaves of infected plants similar to those of wild-type PSV-P were observed. The presence of the virus was confirmed by means of RT-PCR and Western blotting. Re-inoculation to N. benthamiana, Phaseolus vulgaris, and Pisum sativum resulted in analogous results. Generation of infectious clones of PSV-P enables studies on virus-host interaction as well as revealing viral genes functions.
构建了花生矮化病毒P株系（PSV-P）的全长cDNA克隆，并通过根癌农杆菌将其导入本氏烟草植株中。利用无缝重组克隆系统，将与PSV三个基因组RNA及卫星RNA对应的cDNA片段克隆到位于花椰菜花叶病毒（CaMV）35S启动子和胭脂碱合酶（NOS）终止子之间的pGreen双元载体中。将质粒导入根癌农杆菌，随后对寄主植物进行浸润接种。在受感染植株的系统叶上观察到了与野生型PSV-P相似的典型症状。通过RT-PCR和蛋白质免疫印迹法证实了病毒的存在。再次接种到本氏烟草、菜豆和豌豆上也得到了类似结果。PSV-P感染性克隆的产生有助于研究病毒与寄主的相互作用以及揭示病毒基因的功能。
