Virus Resistance Mediated By Replicase Gene Of Potato Virus Y

Potyviruses (genus Potyvirus, family Potyviridae) comprise the largest group of plant-infecting viruses that cause serious damage to crops such as potato and tobacco worldwide. Potato virus Y (PVY) and Tobacco etch virus (TEV) are members of the Potyvirus genus. PVY has many variants and new variants will continuously emerge from the interaction of host and pathogen and the creation of new resistant varieties. A new powerful type of resistance, based upon the presence of RNA, known as RNA-mediated virus resistance (RMVR), was characterized by a high level of resistance that was not easily overcome by a high dosage of inoculums. Once it was established, it could be maintained throughout the life-span of plant. A disadvantage of RNA-mediated resistance, however, is the high sequence specificity. This resistance is effective only against viruses with a high sequence homology to the transgene. In previous studies, the common method was to construct different expression cassettes or the same expression cassettes with different plant viral coat protein genes to make transgenic plants resistant to multiple viruses by expressing viral coat protein genes. We showed previously that coat protein (CP) gene of Potato virus Y (tobacco veinal necrosis strain, PVYN) could confer PVYN resistance to tobacco (NC89) plants and we have proved that this resistance is RNA-mediated resistance. And systematically studies showed that transgenic plants by the designing of hpRNA (hairpin RNA) with 3′and 5′end fragments of PVY-CP gene obtained different proportion resistant ratio. Based on the problem in crops production and our previous research, in the first part of our study, we designed hairpin RNA constructs based on five conserved regions of the NIb gene of Potyviruses and generated transgenic plants, and our objectives are to make clear the lowest homology to produce resistance and determine the feasibility of breeding multiple viruses resistance transgenic plants with the strategy of RMVR. The result will contribute to elucidate the mechanism of RNA interfering, and offer a new strategy of cultivating multi-resistance plant. In the second part of our study we use five cDNA fragments derived from different position of PVY-SD5 NIb gene to generate hpRNAs, and we want to dissert if this kinds of tansgene construction could make transgenic plant have different resistant ratio. The result will contribute to the selection of the target sequence and successfully apply the strategy of RMVR, which has an important theory and application value. The main results and conclusions presented in this thesis are as follows:According to the full-length cDNA sequences of PVY-SD1 NIb (1557bp), the specific primers of conserved sequences were designed. The PVY-SD1 NIb gene was served as templates cloning the conserved sequences. The purified PCR products were digested by XbaI/BamHI and SacI/KpnI, respectively, and then these fragments were ligated in vitro by T4 DNA ligase. The ligated products were transferred into E.coli DH5αby heat-shock. PCR and double enzymes digestion demonstrated that we constructed the recombinant plant expression vectors PRIR-Ⅰ, PRIR-Ⅱ, PRIR-Ⅲ, PRIR-Ⅳand PRIR-Ⅴ, successfully. The five recombinant plant expression vectors were transferred into Agrobacterium tumfaciens LBA4404. PRIR-Ⅰ, PRIR-Ⅱ, PRIR-Ⅲ, PRIR-Ⅳand PRIR-Ⅴwere 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 168, 150, 166, 55 and 170 T0 generation transgenic plants with PRIR-Ⅰ, PRIR-Ⅱ, PRIR-Ⅲ, PRIR-Ⅳand PRIR-Ⅴ, respectively. The transgenic tobacco plants transformed with PRIR-I, PRIR-II, PRIR-III, PRIR-IV, or PRIR-V were inoculated with PVY-SD1, PVY-SD4, PVY-SD5, or TEV-SD1 at the third or fourth leaf stage. The PVY-SD1 isolate induced the typical symptom of viral infection on both non-transformed tobacco and vector control plants 20 days after the inoculation. The ratio of resistant transgenic plants obtained for the five different transgenic lines were 26.2%, 22.7%, 36.4%, 20.3%, and 21.7%, respectively. For PVY-SD5, except for the transgenic tobacco containing PRIR-IV, the four other transgenic tobacco lines (PRIR-I, II, III, V) obtained resistance ratios of 2.4%, 3.0%, 15.9%, and 4.3%; all inoculated transgenic tobacco were susceptible to PVY-SD4 and TEV-SD1. In the case of the non-transformed tobacco, all of the isolates induced severe mosaic and/or necrosis of vein symptoms within 20 days of inoculation. The results indicated that RNA-mediated virus resistance is a sequence-dependent RNA degradation pathway, but there is no perfectly positive correlation between the percentage of resistant transgenic plants, the identity of the transgene, and the target sequence. In order to analyze the correlation between the copy number of transgene and resistance, the resistant transgenic plants and susceptible transgenic plants were choose for Southern blot analysis. These results verified that the foreign genes had been integrated into the plant genome, and no correlation was found between number of insertions and type of resistance. 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.According to the full-length cDNA sequences of PVY-SD5 NIb (1557bp), the specific primers of five sequences derived from different position were designed. CDNA fragments of 51bp derived from 5′end (nt1-51), 5′end (nt364-414), intermediate (nt753 -803), 3′end (nt1143-1193) and 3′end (nt1507-1557) were amplified by polymerase chain reaction (PCR). The purified PCR products were digested by XbaI/BamHI and SacI/KpnI, respectively, and then these fragments were ligated in vitro by T4DNA ligase. The ligated products were transferred into E.coli DH5αby heat-shock. PCR and double enzymes digestion demonstrated that we constructed the recombinant plant expression vectors pRIR-NIbⅠ, pRIR-NIbⅡ, pRIR-NIbⅢ, pRIR-NIbⅣand pRIR-NIbⅤ, successfully. The five recombinant plant expression vectors were transferred into Agrobacterium tumfaciens LBA4404. pRIR-NIbⅠ, pRIR-NIbⅡ, pRIR-NIbⅢ, pRIR-NIbⅣand pRIR-NIbⅤwere 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 obtained61, 58, 64, 67 and 56 T0 generation transgenic plants with pRIR-NIbⅠ, pRIR-NIbⅡ, pRIR-NIbⅢ, pRIR-NIbⅣand pRIR-NIbⅤ, respectively.After asexual propagation, the transgenic plants transformed with pRIR-NIbⅠ, pRIR-NIbⅡ, pRIR-NIbⅢ, pRIR-NIbⅣand pRIR-NIbⅤwere analyzed for resistance to PVY-SD5. The results of virus challenge test showed that transgenic plants derived from different regions of NIb gene displayed different degrees of PVY resistance. We obtained resistance ratios of 31.15%,10.34%,51.56%,67.16% and 16.07%, respectively. 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 indicated that RNA silencing was induced.