Study On Cloning,Expression And Genetic Transformation Of Pokeweed Antiviral Protein Gene

The total RNA was isolated from pokeweed (Phytolacca americana) leaves using the method of guanidine isothiocyante and used as template to amplify the total length and deleted mutant pokeweed antiviral protein (PAP) gene by RT-PCR and then the PAP gene was cloned into pGEM-T vector. The sequencing results showed that PAP gene had 99.9% identity comparing with the PAP gene nucleotide sequence reported by Lin et al (1991). The IPTG-inducible expression vector containing the PAP gene was constructed and transferred into E. coli BL21(DE3)-plysS. The specific protein was produced after inducing with 0. 4mM IPTG and its molecular weight was 26Ku. Double diffusion on agar plate and western blotting test results showed that the expression product in E. coli was high identity to PAP extracted by Dr. Philippe Dulieu from pokeweed leaves in France. These revealed that PAP gene has been actually achieved and exactly expressed in E. coli.The deleted mutant PAP gene was also cloned into plant expression vector pBI121 and then the recombinant vector was introduced into Agrobacterium strain LBA4404. After that the vector was transformed into tobacco using leaf-disc method mediated by the Agrobacterium tumefaciens and transgenic tobacco plants were obtained through the screening with kanamycin. The transgenic tobacco plants could delay TMV infection for about 2 months comparing with non-transgenic tobacco plants.The PAP gene was also transformed into wheat by pollen tube pathway-mediated and 320 grains of transgenic wheat was harvested. The seedlings of the seeds were inoculated with the GAV isolate of Barley yellow dwarf virus (BYDV) by Schizaphis graminum (Rondani) and the results revealed that 33 wheat seedlings did not produce the symptom or the symptom was very mild. Of the 33 seedlings there were 7 seedlings containing PAP gene detected with PCR and Southern-blot, transformation- rate was 2.2%. This developed a new material for the wheat virus-resistance breeding.In order to breed maize dwarf mosaic disease梤esistance maize species, the PAP gene was introduced into maize inbred strains and hybrid strains mediated by pollen and particle bombardment,10 transgenic maize plants were achieved and the PAP gene was indeed integrated into the maize genome by analysis of molecular biotechnological methods. The PAP gene in one of the transgenic maize plants had been transcription and translated, the ability of the virus resistance is undergoing.Verticillium dahliae is one of the most serious pathogen of the cotton, because PAP not only has a perfect anti-virus ability, but also shows a fine resistance to fungi, so the PAP gene was transformed into cotton using micro-injection. Green fluorescent protein (GFP) gene was conjugated to the 3' end of the PAP gene in order to screen easily of the transgenic cotton plants. The combined gene was cloned into plant expression vector pBI121 and then transformed. About 5000 seeds of the transgenic cotton were obtained and the some seedlings of the transgenic cotton could give a bright green fluorescence in the dark condition when the cotton seedlings were irradiated with ultraviolet rays. This result reflected that the GFP gene was expressed in the transgenic cotton; dot-blot result also demonstrated that PAP gene was exactly transformed into the cotton. The capacity of the Verticillium dahliae-resistance is being analyzed.The deleted mutant PAP gene was also cloned into yeast secreted expression pPIC9K vector to form pPIC9K~3, then the vector was transferred into Pachia pastoris GS115 strain. The specific expression protein was secreted into the medium after inducing with methanol and the protein amount reached about 50-60 U g per millilitre measured by UV-absorbed methods in the supernatant of the medium via high density fermentation. SDS-PAGE results showed that there was one protein band in the gel which molecular weight was about 34Ku . The protein could specific react with France PAP antiserum in Western-blotting. Activity tests revealed t...