| Based on the obtained TO transgenic tobacco plants expressing dsRNA of partial Cucumber mosaic virus (CMV) replicase gene or partial Tobacco mosaic virus (TMV) movement protein gene, the successive T1-T4 generations of transgenic tobacco plants were analyzed for virus resistance and the mechanism for resistance was also studied.Seeds were randomly selected from resistant and susceptible TO progenies expressing dsRNA of CMV partialΔRep gene, and the successive T1 seedlings were analyzed by Southern blot. The transgene copy numbers varied among the progenies of different resistance lines, and there was no co-relationship between viral resistance level and transgene copy number. The T1 seedlings derived from seeds of immune TO lines segregated with both resistant and susceptible after CMV inoculation with ratio of 1:1-1:3 (immune:susceptible). The T1 generation seeds were obtained by self-pollination from the four resistant lines and one susceptible line, and then the successive T2 seedlings of each line were inoculated with CMV at four-leaf time. As suggested, T2 progenies derived from resistant T1 lines almost kept the same resistance level as the parent lines. But 100% of the T2 progenies derived from the susceptible T1 line and wild tobacco plants were infected by CMV, indicating that the viral resistance level of T2 progenies was stable. Southern blot analyses showed that T2 lines Rep15-1-1 and Rep15-1-61 carried single copy of trangene. The T3 generation seeds were obtained from immune T3 lines Repl5-1-1-15, Rep15-1-1-26 and Rep15-30-23-27 by self-pollination, and the resulting T4 progenies were tested for their resistance to CMV. No virus was detected in inoculated T4 progenies derived from Rep15-1-1-15, Rep15-1-1-26. The northern blot hybridization analyses of T4 progenies derived from Rep15-1-1-15 confirmed that only limited transcript of the transgene could be detected before and after virus inoculation. Whereas the accumulation level of the viral genomic RNA was very high in CMV infected non-transgenic tobacco plants. The northern hybridization analyses also showed that siRNAs of both 21 nts and 24 nts could be identified in inoculated and non-inoculated transgenic plants with similar accumulation levels, but no siRNA was detected in CMV inoculated or un-inoculated non-transgenic tobacco plants. The above findings indicated that the resistance was resulted from RNA silencing. The virus RNA and siRNA of T4 progenies cultured at 24℃and 15℃were compared after CMV inoculation. At 15℃, the virus RNA was low beyond a detected level and siRNA was accumulated at a similar level as at 24℃, demonstrating that the transgenic tobacco plants still keep resistance at low temperature.Seeds were randomly selected from resistant and susceptible TO progenies expressing dsRNA of TMV partial MP gene, and then the successive T1 seedlings were analyzed by Southern blot. The transgene copy numbers varied among the progenies of different resistance lines, and there was no co-relationship between viral resistance level and transgene copy number. The T1 seedlings derived from seeds of immune TO lines segregated with both resistant and susceptible after TMV inoculation with ratio of 1:1-1:3 (immune:susceptible). Resistance test showed T2 seedlings were resistant to TMV infection. Southern blot analyses showed that T2 lines MP16-17-14, MP16-17-29 and MP53-52-7 carried single copy of transgene, and T4 seedlings derived from MP16-17-14, MP16-17-29 and MP53-52-7 were resistant to TMV infection. The northern blot hybridization analyses of T4 progenies derived from MP 16-17-29-9 confirmed that only limited transcript of the transgene could be detected before and after virus inoculation, but high accumulation level of the viral genomic RNA was detected in TMV infected non-transgenic tobacco plants. The northern hybridization analyses also showed that siRNAs of both 21 nts and 24 nts could be identified in inoculated and non-inoculated transgenic plants with similar accumulation levels, but no siRNA was detected in TMV inoculated or un-inoculated non-transgenic tobacco plants. The above findings indicated that the resistance was resulted from RNA silencing. The T4 transgenic tobacco plants were proven to keep the resistance to TMV at 15℃.Agrobacterium expressing CMV partial Rep (i/r) sequences was co-cultured with tomato leaf disk, and then transgenic tomato lines were obtained after Kanamysin screening. The specific DNA fragment was amplified in randomly selected transgenic tomato lines by PCR, and specific signal was also identified by dot-blot hybridization. The resistance to CMV infection of the transgenic tomato plants expressing dsRNA of CMV partial Rep gene was tested.
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