| The viral diseases have caused severe damage to tobacco by means of quality and yield reducing. However, the species of virus infecting tobacco are still unidentified, and the preventing methods are also inadequate. Thus, our study focused on surveying the virus infected tobacco, and evaluating the preventing potential of the novel RNAi technology.1. According to our survey, Tobacco Mosaic virus (TMV), Cucumber Mosaic Virus (CMV) and Potato Virus Y(PVY) are three most spreading viruses in tobacco in Hunan province. Samples with viral symptoms collected during2011-2013from Xiang-xi, Yongzhou, Hengyang, Changsha of Hunan province were tested, which showed PVY was highest detected in all samples, with positive rate of80.8%, furthermore, the composite infection with these three viruses was common phenomenon in tobacco in Hunan, with positive rate of45.6%2. EFla protein is one of viral transmitted-related protein in Aphid (Myzus persicae), so it is postulated the ratio of viral transmitting would reduce if this protein function was interfered by RNAi method. To this end, the full length of EFla gene of Aphid was sequenced by RT-PCR and Sango sequencing. Two small reverse complementary fragments (named EFla-1and EFla-2) for RNAi were cloned from the conserved region of EFla gene, then, transferred into plasmid vector LITMUS-28for prokaryotic expressing. The obtained fragment containing the EFla-1and EFla-2was thereafter transferred into eukaryotic plasmid vector pFGC5941for plant expressing.3. For evaluating the preventing potential of viral disease in tobacco by RNAi method, two dsRNA fragment of RNA2and MP genes of CMV, which expressed by E.coli, were applied in tobacco. The results showed that these two dsRNA reagent could effectively prevent tobacco viral disease, with control efficiency up to71.4%, as well as improved growth condition of tobacco. These results also provided the promising protocol for preventing and controlling plant viral disease by RNAi method. |
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