Introduction Of Thaumatin Gene Into Tobacco (Nicotiana Tabacum L.) Through Agrobacterium-miedated Genetic Transformation

Sweet protein Thaumatin has great economic value because thaumatin is a no caloric sweetener that has many advantages such as high sweet intensity, low calories, no toxicity and so on. However, this protein can't meet people's dem--ands because of lacking of resources. Therefore, in this paper, the recombinant plant expression vector pBIi2i-th was constructed successfully and introduced into Agrobaterium tumefaciens LBA4404. Subsequently thaumatin gene was transformed to tobacco through Agrobaterium-mediated system. And transgenic plants were confirmed by a series of examinations. The results were as following:1. Construction and identifcation of recombinant plant expression vector pBI!2i-thBy DNA recombination technology, sweet protein thaumatin gene was cloned into plant expression vector pBIi2i. Recombinant plasmid pBIi2i-th was constructed successfully by enzyme cutting and electrophoresis.2. Introduction of pBI121-th plasmid into Agrobacterium tumefaciens . The pBI121-th was introduced into Agrobacterium LBAncn by direct transformation .By screening in the medium containing kanamycin , it was confirmed that the result was successful .3. Regeneration system of plants.The regeneration system of tobacco was established. The adventitious shoots were induced from leaf explants of tobacco based on MS basal medium supple--mented with 2.0mg/L 6-BA and 0.3mg/L NAA . Then the regenerated plants were rooted on MS medium containing 0.3mg/L NAA. And the frequency was about 65.6%, 95% respectively.4. Transformation and selection of plants.Leaf explants had been pre-cultured for 2 days, then immersed in Agroba--cterium suspension for 5~6minutes. The co-cultivation had been carried out for 2~3 days. After that, the transformants were obtained by transferring explants to selection medium containing 75mg/L kanamycin and rooting medium contain--ing 75mg/L, 100mg/L kanamycin .And its rooted frequency was 82.5%.5. Detection of transgenic plants.The PCR assay of kan-resistant plants showed that the target gene had been integrated into tobacco accompanying with npt II gene. The frequency of transf--ormation was 31.3%.