Construction Of A Plant Binary Expression Vector With Pti5-VP16 Gene And Studies On Transgenic Tobacco Plant

颬ti5 gene production was transcription factor that controls the expression of PRs gene. Pti5-VP16 gene was subcloned to plasmid pUCIS and the recombinant plasmid pUCHl was obtained.The inserted gene was sequenced and analysed with molecular biology software DNASIS.Further Pti5-VP16 gene was cloned to plasmid pBI121.The plasmid pBI121UCHl carrying Pti5-VP16 gene under control of the cauliflower mosaic virus 35s promoter was constructed and transformed into Agrobactrium tumefaciens EHA105 by the freeze-thaw method.Tobacco (SRI ) explants from leaf were cocultivated with Agrobacterium tumefaciens EHA105 with the plasmid pBI121UCHl.Inoculated explants were cultured successively on cocultive media,selective media and root growth media.27 Km-resistant tobacco plants were obtained, all the plants were transferred to soil survive.The approaches of PCR,Southern blotting were used to identify the selected resistant plants.All 27 Km-resistant plants were positive by PCR.The plants were then analyzed by Southern blotting,using a -32P labeled PH5-VP16 gene as a probe.Hybridization signals were observed.The Pseudomonas sryingae pv tabaci was used to identify the disease resistance of transgenic tobacco plants.The result showed that the ability to disease resistance was improved in the transgenic tobacco plants.As the transcription of the PRs gene could be enhanced by Pti5 gene production,the ability of disease resistance of transgenic plants was also increased.