Production Of Transgenic Tobacco Plants With Resistance To Two Viruses Via RNA-mediated Virus Resistance

Plant viruses are major pathogens in the cultivation of many economic crops throughout the world,causing serious yield losses every year. The losses of crops such as potato and tobacco, were up to 80%,especially, the potato virus Y veinal necrosis strain(PVYN) often caused host plant early death and yieldreduction. If potato was infected by potato virus X (PVX) singly, the potato yield would decrease 10~20%.However, if coinfected with other viruses in the field, the PVX frequently made destructive losses,especially when coinfected with PVY. A new powerful type of resistance, based upon the presenceof RNA, known as RNA-mediated virus resistance(RMVR), was characterized by a high levelof resistance that was not easily overcome by a high dosage of inoculum. Once it wasestablished, it could be maintained throughout the life-span of plant. Since the protein is notnecessary for conferring resistance, untranslatable constructs can be used, avoiding theaccumulation of any foreign protein and eliminating the risks involved withtransencapsidation. A disadvantage of RNA-mediated resistance, however, is the highsequence specificity. This resistance is effective only against viruses with a high sequencehomology to the transgene. We showed previously that the full length of untranslatable coatprotein (CP) gene and different length fragment of PVYN-CP could confer PVYN resistance totransgenic tobacco plants, and we have proved that this resistance is RNA-mediated.Additionally, we obtained different proportion resistant transgenic plants by the designing ofIR(inverted repeat) and DR(directed repeat) with 3′ and 5′ end 200bp fragment of PVYN-CPgene respectively. Higher proportion of resistant transgenic plants transformed with IR wasobtained than that of transformed with DR. Based on the problem in crops production and our previous research, in this study, weconstructed chimeric gene with the combination of different length and order of untranslatablePVX-CP and PVYN-CP. Then we constructed plant expression vectors with these chimericgenes. Furthermore, we constructed plant expression vectors of dsRNA and Hairpin based onpFGC5941 and pROKII respectively, which were constructed with the 200bp fragments of 3′end of PVX-CP and PVYN-CP in different order. In this study, our objectives are to determinethe feasibility of breeding multiple viruses resistance transgenic plants with the strategy ofRMVR.The main results and conclusions presented in this thesis are as follows: 1. The study on the resistance of transgenic tobacco conferred by chimeric transgenesconstructed with different length of untranslatable PVYN-CP gene and PVX-CP gene. 4山 东 农 业 大 学 博 士 学 位 论 文 1.1 According to the full-length cDNA sequences of PVYN-CP(804bp) andPVX-CP(714bp), the specific primers of untranslatable full-length and 3′ gene fragments weredesigned. The PVYN-CP gene and PVX-CP gene were served as templates cloning the variouslength genes. The purified PCR products were digested by BamHI and XbaI respectively,these fragments were ligated in vitro by T4 DNA ligase. The ligated products were digestedby two corresponding restriction enzymes then cloned into pUC19 that had been cut with thesame restriction enzymes and transferred into E.coli DH5α by heat-shock. After screening therecombinant colonies, we cloned the chimeric genes XY, YX, fXY, fYX successfully. Thegenes proved to be correct by DNA sequence analysis. The chimeric genes excised from thedifferent recombinant cloning vectors were inserted into plant expression vector (pROKII)that had been cut with the same restriction enzymes. PCR and double enzymes digestiondemonstrated that we constructed the recombinant plant expression vectors pROKXY,pROKYX, pROKfXY and pROKfYX successfully. 1.2 The four recombinant plant expression vectors were transferred into Agrobacteriumtumfaciens LBA4404. pROKXY, pROKYX, pROKfXY and pROKfYX were introduced...