Construction Of Expression Vectotr Of Vitamin E Synthetic Gene Of Artemisia Sphaerocephala Krasch And Genetic Transformation Of Tobacoo

Vitamin E is an important class of lipid-soluble compounds with antioxidant activities which were synthesized in photosynthetic organs of higher plants and cyanobacteria. Homogentisate phytyltransferase (HPT) and y-tocopherol methyltransferase(γ-TMT) are two key enzymes, which played an important role in the synthesis process of nature vitamin E.Artemisia sphaerocephala Krasch is an important subshrub growing in desert regions in northern China. It has characters of drought tolerance and sand fixation. The seeds are rich in nutrients, especially high contents of vitamin E, with concentration up to 2.8×103 mg/kg. Artemisia sphaerocephala Krasch is a good resource with valuable functions in research and application. In this study, Artemisia sphaerocephala krasch were used as materail. Bivalent expression vector pBI121-HPT&TMT was constructed using the cDNA cloned in our laboratory. Then, agrobacterium-mediated method was used to infect tobacoo NC89.The main results were as follows:1. Specific primers were designed according to the gene sequences of HPT and y-TMT published in genbank. HPT and γ-TMT genes were isolated from Artemisia sphaerocephala Krasch using RT-PCR method.2. After restriction analysis, pBI121-TMT expression vector was constructed., pBI121-G-HPT was constructedusing pBI121-G.3. Two expression vectors were digested to get two fragments interested. Then, two fragments were connected by ligase. Finally, bivalent expression vector pBI121-HPT&TMT was constructed successfully.4. The bivalent expression vector pBI121-HPT&TMT was transformed into Agrobacterium tumefaciens GV3301, and then transformed into tabacco. The positive tobacco transformants growing on MS medium were selected by spectinomycin.