Study on Transformation of Papaya Ringspot Virus CI and NIB Gene Inverted Repeats Experssion Vector by Agrobacterium Tumefaciens
|School||Huazhong Agricultural University|
|Keywords||CI gene NIB gene Nicotiana tabacum Linn. Carica papaya Linn. hpRNA Genetic transformation|
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 our study, the target genes are the movement protein gene（CI） and replicase gene（NIB） of PRSV. The inverted repeats expression vectors with CI gene and NIB gene were constructed. The recipient materials of agrobacterium-mediated transformed experiment were tobacco leaf discs, papaya zygotic embryos, papaya embryogenic callus and the Arabidopsis inflorescence. The transformed tobaccos and the resistance Arabidopsis were obtained, and we also discussed the problems on the transformation of tobaccos, papaya and Arabidopsis. Morever, we analyzed the protocols of transformation detection.The main results of this study were as follows:（1） Using the agrobacterium carrying pHellsgate-CIIR transformed tobaccos, 100 strains of resistant tobaccos were obtained. 62 strains were analyzed with PCR detection. 14 strains can amplify the 660bp fragment of CI gene, the positive rate was 22.5%.The southern blot were performed to transformed tobaccos which were positive according to the PCR analysis. The positive control was the PCR product which was extracted from agarose gel of pHellsgate-CIIR. The negative control was the genomic DNA of untransformed tobaccos. The result showed that there was no hybridization signal in the transformed tobaccos.The transformed tobaccos were inoculated with PRSV by using the mechanical inoculation method. The untransformed tobaccos were inoculated with the PBS buffer or PRSV. Meanwhile, the untransformed tobaccos were not inoculated with PBS buffer and PRSV as control. After one week, the leaves of the untransformed tobaccos with PRSV and the transformed tobaccos with PRSV both became etiolation or shrinkage. While the untransformed tobaccos not inoculating PRSV grew well. Up to now, there was no virus transmission to other leaves. As to the virus-resistance of the transformed tobaccos, this result awaits the further observation and the molecular detection.（2） Using the agrobacterium carrying pHellsgate-NibIR transformed tobaccos, 20 strains of resistant tobaccos were obtained. 13 strains were analyzed with PCR detection. One strain can amplify the 740bp fragment of NIB gene, and the positive rate was 7.7%.（3） Using the agrobacterium carrying pHellsgate-NibIR transformed Arabidopsis, the offspring seeds were screened in MS medium containing 50mg/L Kan. The result showed that the leaves of Kan-resistance Arabidopsis were totally extension and green. In contrast, the leaves of Arabidopsis which were not resistant to Kan were shrinking and yellow. A few of resistance Arabidopsis were obtained, but the resistance to virus still needs the further detection.