| RNA-mediated resistance is an effective way to obtain virus resistant plants and has attracted many researchers' attention because of its advantage of high bio-safety and long duration. RNA-mediated virus resistance is known as pathogen-derived resistance (PDR) and is one kind of posttranscriptional gene silencing (PTGS). PTGS can be induced by transgenes or viral infection and triggers the degradation of RNAs with homology or complemetarity to the foreign genes' transcript or viral RNA. The signal leading to the degradation of specific RNA can be transmitted. However results from different systems have thrown this simple model into disarray, especially for PTGS signaling. In this research project, tobacco plants transformed with untranslatable PVY CP gene were used to examine the relationship between transgene integration patterns and RNA-mediated resistance more directly . Meanwhile we also investigated the transmission of RNA-mediated resistance and PVY movement on resistant plants and susceptible plants. The main results are as the following: 1. The copy number of foreign genes was always different when then genomic DNA was digested by different restriction endonuclease .The copy number by Hindâ…¢was always more than that by EcoRI in tobacco. When foreign genes were integrated in multiple copies, Southern blot analysis can't reflect the real copy number because the fragments generated are of the same size. In order to get accurate results, we digested genomic DNA with the endonuclease that could produce the most copies and copy number should be determined according to X-ray radioautography.2. Southern blot analyses of T1 plants, which acquired different resistance, indicated that the resistance was related to copy number. The resistant plants contained 3-5 copies while the susceptible plants contained 2-3 copies. But M4-3, M7-1 seem to be the only exception, which, though having high copy numbers, were lack of tandem sequences.Hindâ…¢ and EcoRI were used to digest genomic DNAs from the progeny T1 transgenic plants with different response to PVY infection respectively. The results of Southern blot analysis indicated that almost all of the highly resistant plants contained repeat foreign sequences, including direct repeat (DR) and inverted repeat (IR). The highly resistant plants always contained both of or one of these constructs. While susceptible transgenic plants contained neither of them. Research from different systems identified dsRNA as a joint of various forms of PTGS. In cases where dsRNA could form directly, the triggering of the PTGS process may be straightforward. Readthrough transcription of loci of foreign gene repeats could produce a self-complementary RNA3. that formed a duplex consequently. Thus the arrangement of foreign genes in the plant genome plays an important role in the establishment of resistance.4. By single graft experiments, we discovered that RNA-mediated resistance couldn't be transmitted from highly resistant rootstocks to susceptible scions in spite of the presence of the foreign in the scions. However RNA-mediated resistance is a kind of systemic reaction. Once it occurs, the whole plant becomes immune to virus. This indicated the existence of resistance signals. It is considered that virus move from cell to cell via the plasmodesmata and move over a long distance via phloem. Thus the block of resistance signal might occur in phloem. Consistent with the antiviral nature of RNA-mediated resistance, many viruses use a counter defensive strategy, encoding proteins that block one or more steps in the RNA-silencing pathway. Helper-component-proteinase (Hc-Pro), a viral suppressor of RNA-silencing encoded by plant potyviruses, plays a role in resistant signal blocking by enlarging the size exclusion limit of the phloem. Otherwise the signal from resistant rootstocks may be too little to induce RNA-mediated resistance in susceptible scions. 5. Double graft experiments indicated that PVY could move through a long stem (about 42cm) of high PVY resistance and led t... |
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