Study On Virus-Resistance Of Papaya Ringspot Virus Through Double-Strand RNA

Papaya ringspot disease(PRSD) is the most destructive virus disease of papaya. Up to now, there was still no efficient methods to completely control this disease. The traditional methods were based mainly on the measures of cultivation. The development of plant genetic engineering provided a new way to the study of plant virus resistance. With the big progress of RNAi, the study on plant virus resistance using inverted repeats expression vector became a hot spot. The plant can acquire resistance to virus when it was transfered the genes deriving from virus into plants hosts.In this study, tobacco, papaya and arabidopsis were used as the plant materials. The main experiment method was agrobacterium mediated gene transformation. The agrobacterium were harboured pHellsgate12-CPIR, which was structured the CP gene inverted repeats. And then we analyzed the virus-resistance of transgenic plants.The main results of this study as follows:(1) The WT and pHellsgate12-CPIR transformed tobaccos were inoculated with PRSV and after 3 days there is no obvious phenotype differences between all the treatments.And then we carried out the PT-PCR expriments. The expression of CP mRNA was detected in the WT tobacco leaves infecting with PRSV, but not detected in the transformed tobacco. It seems that the transformed tobacco induced the RNAi process to resist the PRSV.(2) We carried out the agrobacterium transient expression expriments in papaya. The agrobacterium contained the pHellsgate12-CPIR vector was injected into the cotyledons.After 4 days the cotyledons were inoculated with PRSV and after 7 days the phenotype of apicillary leaves were observed.We could see that the apicillary leaves displaying the phenomenon of lesion,wrinkle and chlorosis in the plants which were not injected with pHellsgate12-CPIR vector. And then we carried out the PT-PCR expriments. The expression of CP mRNA was detected in the papaya leaves infecting with PRSV, but not detected in the papaya which was njected with pHellsgate12-CPIR vector.(3) We used the flora dip to transform the arabidopsis with pHellsgate12-CPIR vector. the T1 offspring seeds were screened on the MS medium containing 75 mg/L Kan.3 of resistance T1 Arabidopsis were obtained,and the genome DNA PCR detection was carried out by CP and NPTâ…¡gene specific primers.All had the target fragments.Then we screened the T2 seeds on the MS medium containing 75 mg/L Kan and we found 2 of them were heterozygotes,the other one is homozygote.6 strains were carried out the PCR analysis randomly by CP and NPTâ…¡gene specific primers.All had the target fragments.The rosette leaves of WT and pHellsgate12-CPIR T2 transformed arabidopsis were inoculated with PRSV on after 7 days the uninoculated rosette leaf turning yellow WT arabidopsis but not in pHellsgate12-CPIR transformed arabidopsis.And then we carried out the PT-PCR expriment.The expression of CP mRNA was detected in the Arabidopsis uninoculated rosette leaves infecting with PRSV, but not detected in the transformed arabidopsis. It seems that the transformed arabidopsis induced the RNAi process to resist the PRSV.