Abstract: 【Objective】 This article was to probe into the feasibility of breeding the transgenic tobacco plants with multi-virus resistance via RNA-mediated virus resistance strategy.【Method】 According to the coat protein (CP) gene nucleotide sequences of Potato virus Y (PVY), Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV), specific primers were designed to sub-clone the 3’ end 100bp cDNA fragment of PVY CP gene, 100bp cDNA fragment of TMV CP gene and 150bp cDNA fragment of CMV CP gene. The three CP gene fragments were used to construct a chimeric cDNA fragment, and were introduced into the plants expressing vector pROKII by inverted repeats, which transcripts into hairpin RNA (hpRNA). There recombinant plant-expression vector pRHPTC was introduced into Agrobacterium tumefaciens LBA4404 by direct transferring method, then the target gene was introduced into tobacco NC89. 【Result】The transformed tissues were selected in the presence of 100mg/L kanamycin and the transgenic plants were confirmed by PCR, 276 transgenic tobacco plants were obtained. Southern blot and Northern blot analysis of transgenic plants demonstrated that the target chimeric DNA were integrated into tobacco genome DNA with different copies and transcribed in almost all the transgenic plants, the RNA accumulation level of the transgenic DNA had evidently different in different transgenic plants. The resistance assay indicated that about 23% transgenic plants were immune to the co-infection with PVY, TMV and CMV. The investigation to the transgenic plant generations showed the multi-virus resistance could stably inherit into next generations. 【Conclusion】These results showed that the virus resistance was RNA-mediated. The stably genetic transgenic tobacco plants with multi-virus resistance can be obtained via RNA-mediated virus resistance strategy

Key words: RNA-mediated virus resistance, hairpin RNA (hpRNA), transgenic tobacco with multi-virus resistance