Studies On Expression Of A Pokeweed Antiviral Protein (PAP) Gene In Pichia Pastoris And Genetic Transformation Of PAP Gene

It has been revealed that Pokeweed Antiviral Protein (PAP) can inhibit the infection of many plant viruses. So it can be used to control plant viral disease. The studies include two parts. In the first part, cultivation conditions such as carbon sources, inoculation ration, methanol concentration, pH, D-biotin concentration, shaking speed and induced time were studied. In further research, the fermentation strategy was optimized by orthogonal operation and a better strategy to produce PAP was obtained. SDS-PAGE results showed that the final PAP concentrations were 45mg/L. Western-blotting showed that the protein could specially react with PAP antiserum. Activity tests revealed that the protein could inhibit TMV infection with high efficiency. In the second part, PAP gene was transferred into Petunia hybrida in order to obtain transgenic plants that can control the infection of some plant viruses. At first the plant expression vector pBI121 with PAP gene was cloned into Agrobacterium strain LBA4404 by freeze-thaw method, and the resulting Agrobacterium transformants were verified by PCR amplification. Then the PAP gene was transferred into Petunia hybrida by leaf-disc method mediated by the Agrobacterium transformants. Transgenic plants were obtained through the screening with kanamycin. PCR amplification confirmed the results. This part of studies accumulated the foundation for the advances of plant gene engineering of P. hybrida.