Study On Control Plant Cucumber Mosaic Virus Through Post-transcriptional Gene Silencing Approach

RNA2 fragment, MP gene fragment of CMV isolate P3613, and CP genefragment of CMV isolate AN were amplified by RT-PCR, respectivelty. Three fragments were cloned into vector pBluescript SK(-). To facilitate the construction of inverted-repeat expression vector of CMV genome, the corresponding restriction enzyme sites of CP gene (Bsp120I), RNA2 fragment (KpnI and Bsp120I) and MP gene (PsyI and Bsp120I) were introduced by PCR, respectively. Two inverted-repeat expression vectors, using CP gene as a space, of CMV RNA2 and MP, namely RNA2-CP-RNA2 and MP-CP-MP, were built up under the control of T7 promotor by conventional molecular cloning technique. Furthermore, the two recombinant plasmids were confirmed by PCR, restriction enzyme digestion and sequencing.The two expected dsRNA fragments were expressed through in vitro transcription. The two plasmids were transformed into E.coli HT115, which is RNase-III deficient, respectively. Moreover, the expression crude of large amount dsRNA were used for treating plants to prevent virus infection. Two types of experiments were designed: the CMV were inoculated before dsRNA treatment or vice versa. The existence of CMV in tobacco were confirmed by RT-PCR of CP gene. No virus was detected in both treatments. The results showed that the bacterial crude can induce plant resistance to corresponding virus.Our results lead a way for plant virus control using dsRNA through PTGS approach. Now we are treating the virus infected plants in greenhouse to find the best condition for using this technique in field in near future.