Study On Genetic Transformation Of Apples (Malus Domestica Borkh, White Winter Pearmain) With α-Farnesene Synthase Gene

α-Farnesene is one of aroma related volatile in fruit,which has been proved to play an important biological effect on the attraction of codling moth, and has been associated with the occurrence of scald which is one of serious disorders of apple and pear fruit during storage. The accumulation ofα-farnesene in the skin of apple fruit during storage appears to be predominantly through classical mevalonate pathway. 3-Hydroxy-3- methylglutaryl CoA reductase (HMGR), farnesyl diphosphate synthase (FPPS) andα-farnesene synthase (AFS) are the rate-limiting enzyme in the mevalonate pathway. AFS is the final, rate-limiting enzyme that converts farnesyl diphosphate (FPP) toα- farnesene in the mevalonic acid (MVA) pathway of terpene biosynthesis.To elucidate function ofα-farnesene synthase gene, sense and anti-sense expression vector, RNAi and PBI-AFS-GFP vectors were constructed, a highly efficient regeneration system and genetic transformation system of apple (Malus domestica Borkh, White Winter Pearmain) estabished. The main results were as follows:1. The sense and anti-sense recombinant plant expression vector were constructed, transfored to Agrobacterium EHA105, and used in genetic transformation system. 2. A double-stranded RNAi plasmid was constructed, the results confirmed by restriction enzymes digestion and sequenced shows that the constructed RNAi plasmid contained the designed structure.3. The cDNA of AFS was cloned, PBI-AFS-GFP eukaryotic expression vector was constructed, transformed into epidermal cells of onion by Agrobacterium tumefaciens; transient expression of AFS-GFP fusion protein was acquired, and the result showed that AFS was localized in the cell membrane.4. Adventitious bud regeneration from the leaves of White Pearmain in vitro with different media and hormones of tissue culture was studied. The results showed that the optimum medium for adventitious bud regeneration was MS+BA 4.0 mg/L + TDZ 1.0 mg/L + IAA 0.8 mg/L+sucrose 30.0 g/L+agar 6.0 g/L. The optimum dark incubation time was 20 d. The optimum rooting medium was 1/2MS +IBA0.7mg/L+NAA0.1mg/L +sucrose30.0g/L+agar 6.0g/L.5. An efficient gene transformation system for apples (Malus domestica Borkh, White Winter Pearmain) was established. It was showed that the optimal dose of kanamycin is 15mg/L for gene transformation, and Cefotaxime at a dose of 100mg/L and carbenicillin at a dose of 100mg/L were the optimum antimicrobial antibiotic doses.6. The sense, RNAi recombinant plant expression vector were transformed into apples (Malus domestica Borkh, White Winter Pearmain); the antisense, RNAi recombinant plant expression vector were transformed into Fertility Pear (Pyrus Communis L).