Identification Of CDNAs Associated With Ascobate Acid Metabolism In Tomato

Vitamin C (L- ascorbic acid, AsA), a hexose lactone, plays an extensive role in plant and most animals. It is becoming increasingly clear that vitamin C is of crucial importance in such processes as ROS detoxification, photosynthesis and photoprotection, cell division and growth. AsA is also a cofactor for some deoxygenase type enzymes involved in the synthesis of a number of other secondary metabolites and ethylene. A major breakthrough in plant AsA biosynthesis and metabolic engineering has been made recently. The technique of RNAi has already been extensively applied in gene function research. This study intends to identify the function of genes associated with AsA metabolism preliminarily using the RNAi technique and expects to obtain tomato materials with high content of vitamin C.Using the BLAST program at database of EST (TIGR), two candidate genes encoding ascorbate peroxidase (APX) and ascorbate oxidase promoter-binding protein (AOBP) were obtained. Then, RNAi vectors were constructed with cDNA fragments by RT-PCR and transgenic plants were obtained via Agrobacterium-mediated transformation method. We verified the kanamycin-resistant plants that transgenes had been integrated into the genome DNA by PCR and Southern blotting hybridization and analyzed the physiological and biochemical characteristics of transgenic plants. The main results were as follows:1. Sequence analysis showed that one candidate gene is TC154675, homologous to APX genes of different species, the other candidate gene is TC155602, homologous to AOBP genes of different species2. RNAi plant expression vectors were constructed with cDNA fragments by RT-PCR and transgenic tomato plants were obtained from genotypes of A57 using the Agrobacterium -mediated transformation method.3. The TDNA had been integrated into the tomato genomes by analysis of Southern blotting hybridization.4. The inheritance fashions of the transgenes in T₁ segregation population were analyzed based on PCR with NPTII primers. And the results showed that NPTII gene inherited as a dominant genetic locus with the segregation ratio 3:1.5. Measurements of the content of AsA, the activities of APX and AOBP of T₁ transgenic plants were performed and the results showed that the contents ofAsA increased and the abilities of enzymes decreased for most of the transgenic plants. And there were significant variations among transgenic plants.