Single ShRNA Mediated Virus-resistance To Potato Virus Y And Tobacco Etch Virus

Potyviruses (genus Potyvirus, family Potyviridae) comprise the largest group of plant-infecting viruses, and there are about 200 determined and possible species. They can infect many kinds of plants including solanaceae, chenopodiaceae, leguminosae, the gourd family and so on. Potato virus Y (PVY) and tobacco etch virus (TEV) are two typical members of Potyviruses. A new powerful type of resistance, based upon the presence of RNA, known as RNA-mediated virus resistance (RMVR), is characterized by a high level of resistance that is not easily overcome by a high dosage of inoculums. Once it is established, it can be maintained throughout the life-span of plants. A disadvantage of RNA-mediated resistance, however, is the high sequence specificity. This resistance is effective only against viruses with a high sequence homogy to the transgene. So the common method is to construct different expression cassettes or the same expression cassettes with different effective plant viral DNA fragments to make transgenic plants resistant to multiple viruses. RNA silence is sequence dependent gene silence,so another strategy of fostering more antiviral transgenic plants by RNA silence is based on relatively conservative sequence among different virus (viral strain) .By comparing PVYN 'and TEV-SD1' genome sequences, we chose nine target mRNA sites with high similar and siRNA featuers, and constructed shRNA expression vectors respectively, then transformed tobacco. We found that it could use single shRNA to obtain the multi-antiviral plants, but the antiviral resistance was completely not positive correlation with hoMogy. There were difference in identification of target sequence by base pairs. The results of the research provide the basises to further illustrate the mechanisms of RNA mediated gene silencing (virus resistance), hunt high similar target sequence and use the strategy to foster multi-antiviral (strain) transgenic plants.The main results and conclusions presented in this thesis are as follows:(1) TEV was first isolated from susceptible tobacco in Shandong Province, China, and was designated as TEV-SD1.We cloned the entire genomic sequence of TEV by 5'RACE and RT-PCR. Sequencing results showed that TEV genome cDNA contains 9414 nucleotides (GenBank Acc. No.: EF470242) and encodes 3053 amino acids.(2) By comparing the sequence hoMogy and the siRNA related standards, we selected nine target sequence with obvious features. Designed and synthetized nine primers, and annealled dsDNA. Insert into the pROKⅡdigested by restriction enzyme BamHⅠand KpnⅠ. The ligated products were transferred into E.coli DH5αby heat-shock. PCR and double enzymes digestion demonstrated that we successfully constructed the recombinant plant expression vectors pR-NIB-P1, pR-NIB-P2, pR-HC-P3, pR-HC-P4, pR-CI-P5, pR-CI-P6, pR-HC-P7, pR-NIB-P8 and pR-VPG-P9.(3) The nine recombinant plant expression vectors were transferred into Agrobacterium tumfaciens EHA105. Instantaneously infect the Nicotiana benthamiana, and inoculated with the PVY isolates, then siRNA hybrid analysis.The results confirmed shRNA could express specific siRNA.(4) The nine recombinant plant expression vectors were transferred into Agrobacterium tumfaciens LBA4404. pR-NIB-P1, pR-NIB-P2, pR-HC-P3, pR-HC-P4, pR-CI-P5, pR-CI-P6, pR-HC-P7, pR-NIB-P8 and pR-VPG-P9 were introduced into tobacco (NC89) plants via Agrobacterium tumfaciens-mediated transformation. The transformed tissues were screened in the presence of kanamycin, and the transgenic plants were confirmed by Kanr screening and PCR. We obtained 86, 106 ,104, 111, 80, 99, 114, 86 and 93 T0 generation transgenic plants with pR-NIB-P1, pR-NIB-P2, pR-HC-P3, pR-HC-P4, pR-CI-P5, pR-CI-P6, pR-HC-P7, pR-NIB-P8 and pR-VPG-P9 respectively.(5) Transgenic plants were manually inoculated with the PVYN isolates. The result was 65.12%, 50.49%, 11.54%, 45.95%, 17.50%, 21.21%, 42.11%, 53.49%, 22.58% respectively. All the transgenic plants resistant to PVYN were asexual extender numerous, then inoculated with the TEV-SD1 isolates. The result is 41.86%, 50.94%, 7.69%, 40.54%, 15.00%, 18.18%, 28.94%, 44.19%, 19.35% respectively. The result illustrates that hoMogy above 85.71% can mediated resistance, but resistance and hoMogy is not completely positive correlation.(6) The total RNA was extracted from the transgenic plant with susceptible or resistant responses. Northern blot analysis showed the levels of transcript accumulation varied among transgenic lines. The RNA accumulation level of resistant plants was lower than that of the susceptible transgenic plants. There was an inverse correlation between the resistance and the amount of RNA accumulation in the transgenic plants. Northern blot analysis of siRNA showed that siRNA were detected in the resistant transgenic plants. The results proved this resistance was RNA-mediated.(7) By analysis of factors influenced siRNA activity, we found that the resistant is mainly dependent on 5 ' sense chain base properties and the position of target mRNA.(8) By comparing the mismatch between the sense chains and TEV-SD1 sequence with the resistance to PVYN and TEV-SD1, we found that the position of base mismatch produced significant influence in disease resistance, too.(9) By the disease situation of T1 generation transgenic tobacco, we found that the generationT1 transgenic tobacco can get high level in the disease-resistant.