| RNA-mediated virus resistance is an evolutionarily conserved eukaryotic adaptive response that lead to the specific degradation of target mRNA in response to cellular exposure to homologous double-stranded RNA (dsRNA) molecules. It is induced by transgenes derived from viral genes and regarded as a potential strategy with application value in plant resistance breeding against viruses. DsRNA has been shown to be the key trigger molecules for RNA silencing in organisms. In the process of degradation of target mRNA, RNA degrading system recognizes the invading viral genome and degrades them with the transgenic RNA.Recent research indicated that dsRNA from a variety of sources, (such as transgenes, RNA viruses, transposons, etc.) had been shown to be the key trigger molecules for RNA silencing in various organisms, there are many kinds of formation among various eukaryotic organisms. In plants, transformation of Inverted Repeat (IR) to hosts is considered as a particularly effective way to induce gene silencing, this IR structure can form hairpin RNA post transcription in vivo. The stem of hpRNA determines the specificity of degrading, and is the key part of triggering gene silencing, both its length and origin have influence on inducing RNA silencing. There were researches showed that the length of loop can influence the formation of hpRNA and its stability, then influence the efficiency of gene silencing. It was considered that short loop in favor of the formation of hpRNA, but there were also researches showed that too short loop affect the stability of IR structure in bacteria and the clone of IR structure. So appropriate stem-loop proportion of hpRNA is considered as an effective way to induce gene silencing.In this study six binary vectors with 4﹕1, 2﹕1, 1﹕1, 1﹕2, 1﹕4 and 1﹕ 8 stem-loop proportion of hpRNA were constructed, respectively. Recombinant binary vectors were then introduced into tobacco (NC89) plants via Agrobacterium tumefaciens mediated transformation system, to compare the effectiveness of the six hpDNAs on the induction of RNA-mediated virus resistance. The results of this study provided useful information for the design of effective hpRNA. The main results presented in this thesis are as follows:1. The inverted repeats DNA sequence of hpRNA derived from 3′ends 50bp of PVYNCP gene, and the spacer sequence of hpRNA derived from different parts of pUC19 to form the binary vector pROK-4﹕1,pROK-2﹕1,pROK-1﹕1,pROK-1﹕2,pROK-1﹕4 and pROK-1﹕8.2. Recombinant binary vectors and pROKII were then introduced into tobacco(NC89) plants via Agrobacterium tumefaciens-mediated transform- ation system. PCR and double digestions also confirmed that all the target genes were transferred into Agrobacterium tumefaciens.3. The transformed tissues were selected in the presence of Kanamycin, and the regenerated plants were screened by PCR. 143, 152, 130, 135,132,126 and 40 plants transformed with pROK-4﹕1,pROK-2﹕1,pROK-1﹕1,pROK-1﹕2,pROK-1﹕4,pROK-1﹕8 and pROKII, respectively, were obtained.4. Resistance tests indicated that hpRNA with different stem-loop proportion had different effect on inducing RNA-mediated gene silencing. Plants transformed with pROK-4﹕1,pROK-2﹕1,pROK-1﹕1,pROK-1﹕2 and pROK-1﹕4 were resistant to PVYN infection, and the proportion of disease resistant transgenic plants was 57.34%,61.84%,60.00%,51.11% and 44.69% respectively;when the proportion was 1﹕8, the proportion of resistant transgenic plants was only 9.52%. The results demonstrated that When the proportion was 4﹕1, 2﹕1 and 1﹕1, hpRNA could effectively induce RNAi。5. In order to analyze the correlation between the copy number of transgene and resistance, the transgenic plants which were susceptible or resistant to PVYN were selected for Southern blot analysis, the results showed all the samples had hybridization signals, and no hybridization signal was observed in negative control. There were no obvious correlation between the copy numbers of transgene and resistance. 6. The total RNA was extracted from the transgenic plants with susceptible or resistant responses to PVYN, Northern blot showed the levels of transcript accumulation varied among transgenic lines. Whereas, the RNA in susceptible plants was accumulated to higher levels than that in the resistant plants. The results revealed an inverse correlation between transgenic transcript accumulation and virus resistance.
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