The Comparative Study Of Rice Stripe Virus CP, NS3, SP And NSvc4 Gene-mediated Virus Resistance

Rice stripe disease caused by RSV is one of the most serious viral diseases in most rice-growing areas of China in recent years. RNA mediated virus resistance is a post transcriptional gene silencing. The latest studies demonstrate that RNA silencing in different species is directly triggered by double strand RNA. In transgenic plants researches, an effective way to shape dsRNA is introduce cDNA with hairpin structure into plants. Four binary vectors were constructed, containing different functional genes hpRNAs. They are transformed into rice plants by Agrobacterium tumefaciens-mediated method. Resistance of transgenic plants to Rice stripe virus (RSV) was evaluated afterwards. We also compare the difference on virus resistance mediated by CP, NS3, SP and NSvc4 genes. Major results are as follows:1.We choose fragment of 3′end of RSV CP, NS3, SP and NSvc4 genes insert into binary expression vector pCAMBIA-1300', containing an intron derived from rice phospholipase D gene(PHD). Inverted-repeat constructs were obtained which contained hpRNA structure with stem length of 400bp and loop length of 383bp.2.Recombinant binary vectors pPHD-CP,pPHD-NS3,pPHD-SP, pPHD-NSvc4 and pCAMBIA-1300' were then introduced into EHA105 by the frozen-throwing method (Rhodes,1988). PCR and double digestions also confirmed that all the target genes were transferred into Agrobacterium tumefaciens.3.The recombinant binary vectors eventually introduced into rice (YJ6) accord to a published method (Seiichi Toki, et al, 1997). Transgenic plants were identified by hygromycin B (Zhang, 2005) resistance and PCR. 68, 70, 57 , 58 and 75 plants transformed with pPHD-CP,pPHD-NS3,pPHD-SP, pPHD-NSvc4 and pCAMBIA-1300' were obtained, respectively.4.Transgenic plants were manually inoculated with the RSV isolates by Laodelphax striatellus. Symptom observation and Resistance tests indicated that hpRNA with different functional genes had different effects on inducing RNA-mediated gene silencing. Plants transformed with pPHD-CP,pPHD-NS3,pPHD-SP and pPHD-NSvc4 were resistant to RSV infection, and the proportion of virus resistant transgenic plants was 32.73%, 30.00%, 38.56% and 31.67%, respectively.5.Northern blot and siRNA analysises showed the transcript accumulation varied among transgenic lines on the level of RNA. There is a negative relation between the resistance and accumulation of target DNA fragments' transcription products; siRNAs can also be detected. The results explain that the resistance is mediated by RNA, which means gene silencing. But the results revealed no correlation between accumulation of siRNA and virus resistance.