Genetic Engineering For Virus Resistance In Plant I Construction Of Plant Expression Vector Of Cucumber Mosaic Virus Replicase Gene And Genetic Transformation II Risk Assessment For Release Of Transgenic Plants Containing Virus Derived Sequence

According to the published RNA2 sequence of Cucumber mosaic virus ( CMV ) Fny, the specific primers were designed. The entire CMV Fny 2a replicase gene (RP) with length of 2.5kb was amplified by polymerase chain reaction (PCR). This PCR product was digested with Nco I and BspH I, and three fragments were obtained. Two fragments which couldn't encode GDD motif were ligated and amplified by PCR. And a 2.2kb product with deletion of GDD motif was obtained. The specific primers were also designed for amplification of a 530bp fragment containing partial replicase gene with GDD motif using CMV PI as template. These three replicase genes of CMV were cloned into pGEM-T Easy Vector. DNA sequence analyses confirmed that the ORFs of the amplicated replicase gene were correct.In order to construct expression vectors, the 2a replicase gene and the 2a replicase genewithout GDD motif were cleavaged from pGEM-T Easy Vector with Sma I and BamR I. The 530bp partial CMV PI 2a replicase gene fragment was also cleavaged from pGEM-T Easy Vector with Sma I and Xba I. These replicase genes were cloned into plant expression vector pBI121. PCR and double digestion confirmed that the recombination plasmids contain the target genes.The three genes in plant expression vectors were transferred into Agrobacterium tumefaciens EHA105 by tri-parental mating method. PCR and double digestion also confirmed that the three of target genes were transferred into Agrobacterium tumefaciens.The transformed tobacco plants with CMV Pl-RP gene were obtained via leaf disc transformation mediated by Agrobacterium tumefaciens. PCR analysis of 10 samples of transformed tobaccos showed CMV Pl-RP gene could be amplified from 9 genomic DNA samples with the CMV Pl-RP gene specific primers. 3 genomic DNA samples from PCR positive plants were selected for Southern blot analysis, the result showed all the sample had hybridization signal using CMV Pl-RP gene probe, and no hybridization signal was observed in control. This indicated the target gene has been integrated into the genomic DNA of tobacco. The total RNA were extracted from the 3 Southern blot positive plants, Northern blot showed all the samples had hybridization signal with CMV Pl-RP gene as probe, and nohybridization signal was observed in control. This result indicated the exogenous CMV Pl-RP gene has been transcribed hi the transformed tobaccos.A transformation system was established for the tomato line B-l, The optimum medium and ratio of hormone (MS+Zeatin 0.5mg/ml+BA 3.0mg/ml+IAA0.2mg/ml) to regenerate buds have been founded. The rate of shoot differentiation has reached 500% (the number of buds /per disc).The transformed tomato plants with 2a replicase gene were also obtained via leaf disc transformation mediated by Agrobacterium tumefaciens. PCR analysis of 10 samples of transformed tomatos showed CMV RP gene could be amplified from 7 genomic DNA samples with the CMV Pl-RP gene specific primers. 3 genomic DNA samples from PCR positive plants were selected for Southern blot analysis, the result showed all the sample had hybridization signal using CMV Pl-RP gene probe, and no hybridization signal was observed in control. This indicated the target gene has been integrated into the genomic DNA of tomato. The total RNA were extracted from the 3 Southern blot positive plants, Northern blot showed two samples had hybridization signal with CMV Pl-RP gene as probe, and no hybridization signal was observed in control. This result indicated the exogenous CMV Fny RNA2 replicase gene has been transcribed in the transformed tomatos.The transgenic tobaccos with CMV Pl-RP gene and tomatos with CMV Fny RNA2 2a replicase gene were inoculated with the strain of CMV PI and CMV RB respectively. Some of the transgenic plants were found susceptible to virus infection, delayed symptoms appearance were found in several transgenic plants and a few transgenic plants showed resistance to CMV. Further experiment is still going on.The transgenic tobaccos with movement proteins of ToMV, TMV and r...