Analysis For The Mechanisms Of Resistance Mediated By Virus Genes And Development Of Transgenic Watermelon Resistant To Virus Infection

In order to study the effects of different gene constructs on post transcriptional gene silencing and pathogen mediated resistance, five plant expression vectors -pS18, pR29, pDS24, pII38, pHI19 which contain the TMV CP gene were transferred into Nicotiana tabaccum cv. NC89 mediated by Agrobacterium tumefaciens (LBA4404(pAL4404)) transformation. Southern blots of To transgenic tobacco plants revealed that all forms of the CP genes were inserted as 1 to 6 copies and that the transformation frequencies were 35% to 60%. RT-PCR results suggested that in most of the To transgenic plants, the TMV CP genes were transcribed normally except for the antisense cp of pDS24. Inoculation trials with purified TMV virus indicated that until flowering, some T2 transgenic plants that were immune to virus challenges had a lower number of repeated copies of CP than other transgenic plants. At the same time I analyzed the methylated levels of some T2 and T3 transgenic plants. The results of these assays showed that the copy number of the target gene correlated with the methylation levels. I also observed that the methylation of the target genes affected the resistance of the transformants. In addition, Northern-blot and RT-PCR results showed that three direct repeats, compared with two direct repeats, triggered a more effective PTGS.To obtain virus-resistant transgenic watermelon plants, a trivalent vector pBWCZ-44 containing the genes of Watermelon mosaic virus (WMV) coat protein and the replicases of Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV) were introduced into plants by Agrobacterium using similar strategies as for tobacco. Integrated genes in the regenerated progeny were identified by PCR and Southern blots. These results revealed that 1.7‰ of the regenerated plants were transformed. Detection of resistance to virus infection in the transgenic plants was carried out in greenhouse and field trials. The transgenic watermelons lines developed different disease phenotypes ranging from susceptible, resistant, immune and late recovery to virus infection. Among these plants, the T3 generation of the BH1-7 line had a relative high level of resistance. This result indicates that new varieties of watermelon resistant to virus infection can be created by transformation with viral genes.