Effect Of Overexpressing Potato Dehydroascorbate Reductase (DHAR) Gene In Tomato

L-ascorbic acid (AsA, vitamin C) is an essential micronutrient and antioxidant for plants and human, and also has a multitude of functions in organisms. But humans lack the ability to synthesize AsA. Therefore, AsA must be required regularly from dietary source, primarily from plants. AsA is involved in the process of plant growth and also plays an important role as an antioxident. AsA protects the plant against oxidative damage by scavenging free radicals and reactive oxygen species. By regulating the experssion of genes involved in plant AsA biosynthesis and metabolism, it is possible to increase the AsA contents of plants and futher imporve the nutritive values and the stress tolerance.Dehydroascorbate reductase (DHAR; EC 1.8.5.1) is the key enzyme of the ascorbate-glutathione cycle (AsA-GSH cycle). DHAR allows the plant to recycle DHA to AsA before it undergoes irreversible spontaneous hydrolysis to 2, 3-diketogulonic acid. In this study, we developed transgenic tomato plants that overexpress two potato DHAR genes respectively. Both the relationship between DHAR genes expression and AsA content and the relationship between the change of DHAR genes expression and the tolerance to environmental stresses in tomatoes were investigated. The main results are as follows:1. The potato DHAR1 and DHAR2 cDNA was introduced into tomato by using Agrobacterium tumefaciens and verified by PCR and Real time PCR.The results indicate that the DHAR1 and DHAR2 has been recombined into tomato genome and sense DHAR1, DHAR2 transgenic tomato plants were obtained.2. Measurements of enzyme activities and AsA contents of transgenic plants regenerated from DHAR1 and DHAR2 sense expression vectors were performed. The results indicated that the DHAR activity, AsA contents and AsA/DHA in both leaves and fruits of DHAR1 transgenic plants are higher than that of the wild type plants. The DHAR activity, AsA contents and AsA/DHA in leaves of DHAR2 transgenic plants are higer than that of the wild type plants. But these values have no significant difference with the wild type plants in fruits, suggest that DHAR2 overexpression do not affect the fruits DHAR activity and AsA contents.3. Both the chlorophyll content and photosynthetic rate are increased in leaves of DHAR1 and DHAR2 transgenic plants. Therefore, the overexpression of DHAR have significant effects on the chlorophyll protection and photosynthetic capacity.4. The resistance to oxidative stresses of transgenic plnats were investigated. The results show that salt tolerance and resistance to methyl viologen (MV) stress are improved by the overexpression of DHAR1 and DHAR2. The transgenic DHAR1 and DHAR2 plants show enhanced stress tolerance in terms of significantly higher germination rate, plant fresh weight, seedling stem length and chlorophyll content under salt stress. Furthermore, the transgenic plants also exhibit less necrosis and higher chlorophyll content in leaves than WT under MV stress. Compaired with WT, higher DHAR activity, AsA content and chlorophyll content and lower electrolyte leakage and MDA content were examined in DHAR1 transgenic plants under low-temperature stress. Overexpressing DHAR1 could increase the low-temperature resistance of tomato plants.