Transformation Of Prephenate Dehydrogenase Gene In Broccoli

Vitamin E (tocopherol) is a kind of lipid-soluble vitamin that has manyadvantages on human health such as antioxidant, preventing senescence, improvinglipid metabolism andblood circulation, promoting wound healing. It is onlysynthesized in plant and photosynthetic bacteria. So human can achieve it from thefood.It is necessary to overproduce in the vegetable by biotechnology. The researchshowed that prephenate dehydrogenase (PDH) is the key enzyme in metabolicpathway of vitamin E biosynthesis.4-Hydroxyphenylpyruvate Dioxygenase (HPPD)catalyzes the formation of honogentisate from hydroxyphenylpyruvate and molecularoxygen and is a new target for herbicide such as diketonitril as also. Honogentisate isthe precursor in metabolic way of plastoquinone and vitamin E.This research was considered to transform into broccoli as a strategy forimproving the content of vitamin E. The specific works are as follows:1. According to the record of NCBI, we designed the primers and cloned PDHgene whose similarity are99.6%.2. According to the record of NCBI, we designed the primers and cloned HPPDgene whose similarity are92.60%.3. The plant binary expression vector pCAMBIA3300-35S-PDH-Tnos-35S-HPPD-Tnos makered by HPPD herbicide-resistant gene has been built. Afterintroducing the PDH gene and HPPD gene (instead of bar gene) into broccoli byAgrobacterium mediated method. The PCR and herbicide-resistant experimentsshowed that the plant binary expression vector had been transformed into plant.4. In this research, we optimized the broccoli agrobacterium mediatedtransformation system. For growth of agrobaetium in the initial select medium, theminimum lethal carbenicillin concentration was500mg/L.20mg/L of DKN wasdetermined as the minimum lethal for selection of resistanttransgenici plants. Thebroccoli agrobacterium mediated transformation system had been determined:OD600=0.4～0.6,3～4minutes for infection,3days for coculture and2weeks for onegeneration in multiplication culture...