Special Origin Of PVY And RBSDV CP Influence The Resistance Of Hairpin RNA Against Specific Virus

RNA silence is an ancient phenomenon existed commonly in Eukaryotes, such as plants, mammals, elegans and fungi, which acts as a highly conservative and sequence-spicific degradation of RNA mechanism against viral infections to preserve the genomic integrality. RNA-mediated virus resistance (RMVR) is a post-transcription gene silencing which target to the viral nucleotide acid fragment. It has become a new developed genetic engineering strategy with the advantage of high resistance (almost immunity), long resistant duration and high biosafety. In previous studies, it has been comfirmed that not all the siRNA using criterion established for potential siRNAs showing the same effection. The exact reason is obscure, maybe attribute to the "position effects". In our studies, plant expression vectors of hairpin constructs was designed based on different regions of50bp targeting the Potato Y virus (PVY) coat protien (CP) gene and generated transgenic plants. For the systematic study, plant expression vectors of hairpin RNA (hpRNA) constructs were designed with5’, middle and3’regions of500bp of the Rice black-streaked dwarf virus (RBSDV) CP gene as targets and transformed into rice, furthermore. Then, we ulteriorly explored the resistance levels mediated by RNAi targeting specific regions and obtained transgenic rice highly resistant against RBSDV. The results mostly contributed to the effective selection of the target sequence and application of the strategy to generate transgenic plants with viral resistance. The main results and conclusions presented in this thesis are as follows:(1)Different origins of PVY CP gene influence the resistance of hairpin expressing plants against PVYThe PVY CP gene (801bp) was divided into16regions with50bp, respectively. For every region, target segments were amplified by PCR. Then two fragments were inverted inserted into plant expression vector pROK II. After selection, the plant expression vectors pROK-CPs (pROK-CP1～pROK-CP8) of hpRNA structure were obtained.The16recombinant plant expression vectors (the other8hpRNA expression vectors were constructed by Jiang Fang) were transferred into Agrobacterium tumfaciens EHA105with the freeze-thaw method, and then transferred into N. benthamiana through Agrobacterium tumfaciens-mediated transient infection. Northern blot analysis revealed that siRNAs were detected in all plants in transient assay. And all16biologically active siRNAs could effectively down-regulate the expression of target mRNA. The hpRNA recombinant plant expression vectors were introduced into LBA4404by freeze-thaw method, then vectors were transformed into tobacco NC89by leaf disc method. After the selection of Kanamycin and detection of PCR, To transgenic plants were obtained. The same detections to the progenies of selfed To plants showed that60,55,70,66,66,48,68and72T1transgenic lines were obtained from transgenic groups with vector pROK-CP1～pROK-CP8.Transgenic plants were manually inoculated with the PVYN isolates (the transgenic plants of8hpRNA expression vectors targeting3’-terminal sequence were obtained by Jiang Fang), and evaluated for their resistance by combined symptom and ELISA detection. The resistance ratios of pROK-CP1-pROK-CP4are0, however, other12transgenic tobacco groups exhibited virus resistance with varying ratios of27.15%,6.27%,67.65%,70.83%,66.01%,61.34%,66.04%,23.59%,11.11%,55.56%,77.78%and67.25%. The results indicated that plants harboring the hpRNAi constructs targeting3’of PVY CP can induce higher virus resistance than those targeting5’ of PVY CP.Northern blot analysis of T1revealed that the trangene had been expressed in the level of RNA, and the transcripts accumulation level of resistant plants was lower than that of the susceptible ones. There was an inverse correlation between the resistance and the amount of RNA accumulation in the transgenic plants. In addition, siRNA were detected in all the transgenic plants The results proved this resistance was RNA-mediated, but there is no obvious correlation between the expression level of sequence-specific siRNAs and virus resistance.Virus resistance assay in the T2generation showed that most of the progenies transgenic plants of resistant plant still exhibited high resistance. The results indicated that siRNA-mediated virus resistance can be stably inherited in T2generation.(2) Different origins of RBSDV CP gene influence the resistance of hairpin expressing plants against RBSDVPlant expression vectors of hpRNA structure with three fragments of500bp targeting different regions from5’, middle and3’of RBSDV CP gene (nt89-588, nt589-1088, nt1089-1588) were constructed, named as RBSDV-CP5’, RBSDV-CPM and RBSDV-CP3’, respectively.The three recombinant plant expression vectors were transferred into Agrobaeterium tumfaciens EHA105with the freeze-thaw method. Then Callus of rice cultivar Zhendao88were transformed mediated by Agrobacterium.19,21and22positive plants were obtained respectively after Basta selection and PCR detection of To generation transgenic plants of RBSDV-CP5’, RBSDV-CPM and RBSDV-CP3’.The self-fertilized seeds from To generation transgenic plants were planted to obtain T1generation lines. Leaf painting of Basta selected positive transgenic plants in T1generation, of which50positive plants were inoculated by Laodelphax striatellus carried RBSDV from Jining, Shandong Province. Resistance to RBSDV detection suggested that the resistance ratioes of T1generations were15.80%,23.81%and27.27%, respectively. Results revealed that virus resistance induced by the hpRNAi constructs targeting3’of RBSDV CP was higher that targeting5’of RBSDV CP.Northern blot analysis of T1generation suggested that the transcripts accumulation level of resistant plants was obviously lower than that of the susceptible ones. In addition, siRNA was detected in transgenic plants. These results proved the resistance was RNA-mediated. However, no significant correlation was observed between the accumulation of siRNA and the level of resistance.