Phytolacca Ribosome Inactivating Plant Cdna Expression Vector Construction And Transformation Of Tobacco Research

The specific primer based on the reported eDNA sequence of Pokeweed Anti-virus Protein (PAP) was designed and synthesized. The PAP cDNA fragment, which include the site of restriction enzyme with BamH I at 5end and KpnI at 3end, was amplified by PCR. At the same time, start codon ATG and stop codon TAA were connected with the PAP cDNA fragment. The plant expression pRPAP vector was constructed by cloning the PAP cDNA fragment to pROKII plasmid, The positive recombinant was identified by directly PCR on colony and analysis of restriction enzyme digestion. The engineered strain EHA 105 (pRPAP), which was used in tobacco transforming, was constructed by directly conducting the pRPAP to A.tumefaciens EHAIO5. The efficiency of transformation has reached 37%. The result showed that the PAP gene was integrated into the genomes of tobacco by PCR analysis and southern blotting. The infection test indicated that transgenic tobacco exhibited the higher resistance against fisngal pathogen Rhizoctonia Solwii.