| In the field of plants antivirus gene engineering, people can obtain stably inherited virus-resistance plants by the way of integrating the virus genes into plant genome. This way can overcome the difficulty of the regular methods of screening plant strains, so it became an efficient way of obtaining the resistance plants on antivirus reseach.With the development of plant antivirus research, some researchers focused on RNA mediated virus resistance. RNA mediated virus resistance may be an ancient and conserved mechanism during the plant evolution.The mRNA formed the dsRNA in plant cells, and then it triggered the process of post-transcriptional gene silencing（PTGS） to degrad the target genes. So it can prevent the process of translation in plants cells.In this study, the plant materials were tobacco, Arabidopsis, papaya. The main experiment method was agrobacterium mediated gene transformation. The agrobacterium were harboured pGA-GFP, which was structured a GFP gene, pHellsgates-CPiR and pHellsgate12-CPIR, which both were structured the CP gene inverted repeats. The main results of this study as follows:（1） The transformed tobacco with pHellsgate12-CPIR obtained 120 resistant strains. 90 strains were carried out the PCR analysis randomly. 26 strains can amplify the target PCR product by using the spcial primer of CP gene （P4, P5）. The positive rate was 28.9%Five strains which can amplify the fragment of CP gene were carried out the PCR analysis randomly by using the spcial primer （P4, PDK）. All of the five strains can amplify the target PCR product of 1700bp.Seven strains transforming with pHellsgate12-CPIR which can amplify the fragment of CP gene were carried out the southern blot randomly. The hybridization probe was 861bp CP gene labeling by alkaline phosphatase. The hybrization signals were observed in all seven strains. This result showed that the CP gene was intergrated to the tobacco genome.The total RNA of strains transforming with pHellsgate12-CPIR was extracted by Trizol protocol. Strains transforming with pHellsgate12-CPIR which can amplify the fragment of CP gene were carried out the northern blot randomly. The hybridization probe was 861bp CP gene labeling byα-32P. The expression of CP mRNA was detected in the papaya leaves infecting with PRSV, but not detected in the transformed tobacco.26 strains tobaccos transforming pHellsgate12-CPIR were inoculated with PRSV by using the mechanical inoculation method. After six weeks, seven strains tobacco were no disease symptoms in every stage of plant development, that is, the transformed tobacco were resistant to PRSV. Four strains tobacco appeared some non-uniform disease spots with the diameter form 0.2 to 1cm. 15 strains tobaccos became etiolation in large area, similar to the wild untransformed tobaccos. After six weeks, the leaves were necrosis seriously.（2） The transformed tobaccos with pGA-GFP obtained 5 resistant strains. One strain can amplify the target PCR product of 750bp by using the spcial primer of GFP gene（G20, G21）. The positive rate was 20%, but the expression of GFP protein can not observed.（3） The transformed tobaccos with pHellsgate8-CPIR obtained 70 resistant strains. We need to be performed by PCR analysis in the furture.（4） Using the agrobacterium carrying pHellsgate8-CPIR transformed Arabidopsis, the offspring seeds were screened on the 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 of detection. |
