The Molecular Action Mechanism Of Cytosinpeptidemycin And Ningnanmycin Against Tobacco Mosaic Virus

Cytosinpeptidemycin(CytoM) is a novel anti-viral agent from Streptomyces ahygroscopicus var. liaoningensis developed by plant virus laboratory of Shenyang Agriculture University, Ningnanmycin(NingNM) is a kind of Cystosine nucleocide type anti-viral agent widely applied in the field, both of the two anti-viral agents have inhibiting effect on the disease caused by tobacco mosaic virus(TMV), but the further molecular anti-viral mechanism of the two agents on TMV remains to be clarified. In this study, the complete genome of tobacco mosaic virus liaoning isolate(TMV-LN) was sequenced and constructed to T7 promoter expression vector pUC18(pUC-TMV-LN); secondly, the probe for TMV positive-strand detection in northern blot analysis was constructed based on pUC-TMV-LN; thirdly, the possible molecular anti-viral mechanisms of CytoM and NingNM on TMV was proposed based on the results of northern blot analysis. The main results and conclusions of this study were as follows:1. The plasmid pUC-TMV-LN with TMV-LN complete genome is constructed successfully, this plasmid provide an important material for constructing virus detection probe and studying the action site of the two anti-viral agents in detail. RNA extracted from purified TMV-LN was reverse-transcripted to cDNA, specific primers based on the TMV full genome from Genbank were designed, PCR products named TMV-Halfl and TMV-Half2 were amplified by using the cDNA as a template. The plasmid pUC-TMV-LN with TMV-LN full genome were constructed by enzyme cutting and ligating to same restriction enzyme cutting site vector. The blasting result of TMV-LN full genome sequence and other TMV isolate in Genbank suggested that TMV-LN is 99% similar to South Korea isolate(AB369276.1). The constructing of this plasmid provide a material for studying CytoM and NingNM anti-viral mechanism and constructing RNA detection probe of positive strand.2. Specific and sensitive DNA and RNA probes detection system were constructed for TMV-LN detection in northern blot analysis. To construct the plasmid that express the RNA and DNA probe, target fragment was amplified by using the specific primer and pUC-TMV-LN as a template, the PCR products were ligated into pUC119 to constructed pUC-TMVPP. Digoxigenin labeled RNA detection probe was expressed in vitro from pUCTMV-PP vector using Dig Northern Starter Kit, meanwhile, DNA probe was constructed by using Dig High Prime DNA Labeling and Detection Starter Kit II as a control. Both of the probes showed high specificity and were suitable for TMV-LN qualitative determination through TMV-LN dot-blot hybridization detection system. Subsequently, further precise detection using Northern blot hybridization system was conducted and the results showed that both RNA probe and DNA probe exhibit high specificity and the RNA probe is suitable for relative quantification of genome RNA and subgenome RNA of TMV-LN. In conclusion, both of the molecular hybridization systems can used in specific detection of TMV-LN, dot-blot hybridization system is suitable for virus qualitative detection on account of convenience, whereas, northern blot hybridization has its advantage in relative quantification of viral genome RNA. The construction of RNA and DNA probes in northern blot analysis provides materials for inhabiting mechanism of CytoM and NingNM on the accumulation of viral genome.3. By using the whole plant and protoplast system, the inhibiting effect of CytoM and NingNM on the positive and negative strand of TMV was studied in detail by using TMV-LN as a inoculating source. In whole plant system, protective and curative effect of CytoM and NingNM was studied by inoculating in different time; the inhibiting effect of CytoM and NingNM on systemic infection of TMV was studied from the accumulation of TMV RNA in inoculated leaf and upper leaf by northern blot analysis. In the tobacco protoplast and tobacco BY2 protoplast system, CytoM diluted at 1/3200 significantly inhibit the accumulation of TMV positive and negative strand, NingNM diluted at 1/400 also inhibit the accumulation of TMV positive and negative strand in the tobacco protoplast and tobacco BY2 protoplast system.