| Potato virus Y (PVY) is one of the most important virus diseases of economic crops worldwide, causing serious yield losses every year. To date, we can say that it is the most potent and efficient strategy in genetic engineering of virus resistance. A new powerful type of resistance, based upon the presence of RNA, became known as RNA-mediated virus resistance and was characterized by a high level of resistance that was not easily overcome by a high inoculum's dose. Once it was established it could be maintained during all the life of plant. Since the protein is not necessary to confer resistance, untranslatable constructs can be used, avoiding the accumulation of any foreign protein and eliminating the risks involved with transencapsidation. 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. RNA-mediated virus resistance is the manifestation of PTGS. The induction of RNA-mediated virus resistance has been shown to depend upon the transgene length or the structure.On the basis of demonstrating that the intact CP gene of potato virus Y necrotic strain(PVYN) could initiate RNA-mediated virus resistance in transgenic tobacco plants. We want to find the minimum length of PVYN-CP cDNA that could initiate RNA-mediated resistance. We also want to know the influence of transgene structure on RNA-mediated resistance. The main results presented in this thesis are as follows:1. The effect of PVYN-CP segment length on RNA-mediated virus resistance1.1 According to the full-length coat protein sequence of potyvirus, the specific primers were designed. Two different segments of CP with length of 417bp and 603bp were amplified by polymerase chain reaction (PCR). The PCR products digested with BamHI and KpnI were ligated with pBSK dealt with the same enzymes and transferred into E.coli DH5α by heat-shock. After screening the recombinant colonies, we cloned cDNA fragments located at the 3′end of the PVYN -CP gene with different length, i.e. CP417 (417bp in length at the 3′end) and CP603 (603bp in length at the 3′end) successfully. DNA sequence analyses confirmed the genes were correct. CP417 and CP603 extracted from the different recombinant cloning vectors were inserted into plant expression vector (pROKⅡ). PCR and double enzymes digestion confirmed that we constructed the recombinant plant expression vectors (pROK-CP417 and pROK-CP603) successfully.1.2 The two recombinant plasmids were transferred into Agrobacterium tumefaciens LBA4404 by direct transferring method. PCR and double digestions also confirmed that all the target genes were transferred into Agrobacterium tumefaciens.1.3 CP417 and CP603 were introduced into tobacco (NC89) plants via Agrobacterium tumefaciens-mediated transformation. The transformed tissues were selected in the presence of 100mg/L kanamycin and the transgenic plants were confirmed by Kanr screening and PCR. We obtained 224 tobacco plants with CP417, 77 tobacco plants with CP603.1.4 The transgenic plants with CP417 and CP603 were inoculated with necrotic strain of PVY, respectively. The result of symptom observation and ELISA detection showed that there were three kinds of responses to PVYN, i.e. Susceptible (S), delayed symptoms (D) or resistant to virus infection (R). The resistant assays showed that the proportion of resistance (immune) plants with CP417,CP603 were about 8.48%,10.4%, respectively. I.e., CP417 and CP603 could effectively initiate high resistance to PVYN infection in transgenic tobacco plants. However, the another result from our lab was that no resistant plants were obtained from the transgenic plants containing the fragment of CP202 (202bp in length at the 3′end) . These results revealed that the minimum length of cDNA that could initiate RNA-mediated resistance would be between 417bp and 202bp at the 3′end of the PVYN -CP gene.1.5 In order to analysis the correlation between the copy number of transgene and resis...
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