Studies On Transformation Of Human Wild-type P53 Tumor Suppression Gene In Tobacco

Mutation in the tumor suppressor p53 gene are turning out to be the most common genetic alteration in human cancer. Wild-type p53 gene play an important role in the control of cell cycle , cell transform, DNA replication and induction of apoptosis. Introducing foreign wild-type p53 gene or p53 protein into tumor cell is a potent therapeutic strategy for human cancer. We expect that the transgenic tobacco plant become a bio-reactor and develop a new way to produce wild-type p53 protein.In this study, the coding sequencing of human wild-type p53 tumor suppressor was ligated with the promoter and terminator, which are expressed specially in plant, finally inserted into the plant expression vector pCAMBIA2301 that contain CaMV35S promoter, neomycin phospho transferase (NPT II ) gene and glucuronidase(GUS) gene between Hind III and EcoR I sites, and a recombinant express plasmid pCAMBIA2301/p53 expressing wild-type p53 in plants was constructed. We used tobacco aseptic seedling leaf as explant, transferred the gene into tobacco by Agrobacterium EHA105 mediation and laid a foundation for scale production of p53 from transgenic plant. The main results are as follows.-1 .Construct a genetic transform system with tobacco aseptic seedling leafdisk as explant and obtain kanamycin resistance by introducing p53 gene into tobacco by way of Agrobacterium tumefaciens mediation.2.Effects of antibiotics, including Ceftriaxone Sodium, Cefoperazone Sodium, Cefaxilin Sodium, Cefazolin Sodium and Cefradine on inhibiting Agrobacterium EHA105 and on induction of calli and bud formation of tobacco were studied .The results showed that Ceftriaxone Sodium was the best choice of the five antibiotics and the optimal concentration was 200mg/L.3.Results of histochemical assay, Southern blotting showed that human wild-type p53 gene was stably integrated into the genome of transgenic tobacco and obtained transgenic tobacco plant with p53 gene.