Studies On Construction Of Plant Expression Vector With Antisense PPO Gene Promoted By Patatin Or Wun 1 Promoter And Genetic Transformation In Potato And Tabacco

Polyphenol Oxidase(PPO)is not only related to anti-disease of potato, but also related to browning potato tuber. Now,it has been become a urgent problem that how to ensure higher PPO activity of leaves in potato breeding, and lower PPO activity to decrease the browning degree of potato tuber .The present study was based on the plant expression vector of pC3-ASPPOty with parties PPO antisense gene sequence to construct a new vector in which CaMV35S promoter was insteaded by Patatin or Wound-inducible Promoter. The PPO activity and browning intensity of transgenic tobacco plants with Patatin or Wun1 promoter were tested, it provided beneficial information for potato breeding. The main results were as follows:1. Two specific primers were designed which were added to two restriction enzyme sites BamHâ… and Salâ… . Two fragments of PCR products, 1.1kb and 1.2kb, were amplified, and then they were inserted into pMD18-T plasmid. Two constructs pMD-P and pMD-W were obtained. After enzyme cutting and calling back, these two fragments were inserted into expression vector PC3-ASPPOty, after PCR testing, the recombinant plasmids were named as pCP-ASPPOty and pCW-ASPPOty. They were then introduced into Agrobacterium strain LBA4404 respectively by direct entering ways. It was evidenced that vectors had been transferred into LBA4404.2. Agorbacterium mediated transformations were carried out for tobacco T12 and potato"GANNONGSHU No.1","GANNONGSHU No.2", using Agrobacterium tumefaciens strains LBA4404+pCP-ASPPOty, LBA4404+pCW-ASPPOty, LBA4404+PC3-ASPPOty and LBA4404+PC3-ASPPOqc. Various explants have been tried. The optimum results showed that the tobacco leaf discs were infected with active Agrobacterium suspension(OD600 reached 0.5)for 5~8 minutes, co-cultivated for 2 days on culture medium. When potato stem fragments were pre-cultivated for 2 days, infected with active Agrobacterium suspension(OD600 reached 0.5)for 5 minutes, co-cultivated for 2 days on culture medium. In kanamycin sensitivity experiment, the results showed that suitable concentrations for selecting the...