烟草花叶病毒及其弱毒疫苗-N14基因组cDNA克隆的构建与侵染性分析

Cloning and Infectivity Analysis of the cDNAs of Tobacco Mosaic Virus (Tomato Strain)and Its Attenuated Virus(N14) Genomes

植物病毒弱毒疫苗在防治植物病毒病害中发挥着重要的作用,但对于其致弱的根本原因和机理仍不十分清楚。弱病毒能够在植物体内生存和繁殖,却不严重危害植物的生长、开花和种子生产,而对外来病毒具有杀灭和防治作用,这种共生和保护宿主的现象是大多数植物弱病毒所具有的共同...

An oligonucletide primer with SP6 transcription promoter and the other primer with a Kpn I site at 5' terminuses were used to prepare the cDNAs of tobacco mosaic virus(TMV,Chinese isolate) and its attenuated virus(N14,nitrite treated mutant,tomato strain)genomic RNAs by RT PCR.The cDNAs were cut into two fragments by Bam HI and first cloned separately then recombined into pUC18 by the restriction endonucleases of Bam HI, Sma I, Hin cⅡ, Pst I, Sph I.The sequences of 5' and 3'terminal nucleotides (<50bp) were consistent with the reported references.The infectious clones(pTMV Cv/pN14)have been obtained and corroborated by in vitro transcription with SP6 RNA polymerase and tobacco infection.Necrotic local lesions were observed in Nicotiana samsun NN induced by the transcripts from both pTMV and pN14 within 3～5 days after inoculation,systemic symptoms showed in single lesion infected Nicotiana tobacum cv. coincided with symptoms of tobaccoes infected by TMV and N14.