Tobacco Mosaic Virus (TMV) Coat Protein Induce Tobacco Against Plant Virus

Plants induced resistance reffered to activate their own resistance mechanism by elicitors. In recent years, the development and using of resistance inducing agents have opened up a new field of plant protection. Using inducer of plant disease resistance is also an ideal alternative to control and prevent plant virus disease.In this study, ozone treated Tobacco mosaic virus (TMV) virions and fusion expressed TMV-CP were obtained to investigate their effects on virus resistance of tobacco against TMV. The main results were:TMV virions was extracted from TMV- infected tobacco. The purified virus was then treated by 6g/L ozone and dialysised to obtain TMV-CP.Total RNA was extracted from TMV-infected tobacco leaves and the full-length CP was amplified by RT-PCR. The specific fragments were cloned into pMD18-T. Sequnence analysis showed that the cloned gene was 99% homologue to that of TMV-U1. Using the expression vector pET-29a, The recombinants pET-29a-CP was obtained and transformed into E.coli BL21(DE3). IPTG (Isopropylβ-D -1-thiogalactopyranoside) induce fusion protein expressed. A 25 kDa fusion protein which showed in the same size as expected was detected by SDS-PAGE, suggesting that the protein was successfully expressed. The solubility of the fusion protein was analyzed by ultrasonic and the result showed that the protein is insoluble.Using phosphate buffer solution (PBS) prepare for fusion expressed TMV-CP.Tobacco plants was treated by ozone-treated TMV-CP and fusion expressed TMV-CP (10μg/mL) respectively. The inoculation of TMV was conducted 3 days after the treatments. A week later, ELISA (Enzyme Linked Immunosorbent Assay, ELISA) was used to detect TMV in treated and control plants. The results showed that: Virus relative concentration (OD) in tobacco plants delt with ozone-treated TMV-CP was 33.6% of that in positive control; the OD of tobacco treated by fusion expressed TMV-CP was 47.5% of that in positive control. The results show that two kinds of TMV-CP could induce tobacco anti-TMV infecting.Enzyme activity of POD, PPO, PAL, SOD, content of chlorophyll and MDA in treated tobacco plants were measured. The results indicated that ozone-treated TMV-CP and fusion expressed TMV-CP could enhance the activity of these defensive enzymes and reduce the accumulation of MDA and decrease of chlorophyll, soluble sugar and proteins in TMV infected tobacco plants. Isozymes spectrum of POD, PPO and pathogenesis-related protein were observed. The results showed that there was one isozymes spectrum an additional PR band which were not found in untreated or healthy plants. These results suggested that both ozone-treated TMV-CP and fusion expressed TMV-CP could induce resistance responses and reduce negative effects of viral infection on plant hosts to some extent.