Cloning Of DHAR CDNA From Apple[Malus Domestica] And Plant Expression Vecter Construction

Ascorbate acts as an important antioxidant in both enzymatic and non-enzymatic reactions in plant cells. For maintenance of the antioxidative activity of ascorbate, its regeneration is necessary. Ascorbate is concomitantly oxidized to monodehydroascorbate. Some monodehydroascorbate radicals are re-reduced in a reaction catalyzed by monodehy- droascorbate reductase, but the remainder undergoes spontaneous dismutation to ascorbate and dehydroascorbate. Dehydroascorbate reductase catalyzes the re-reduction of DHA to ascorbate by glutathione. Thus, dehydroascorbate reductase is critical for protection of cellular components against oxidative injury.In this paper, we report a cDNA encoding the apple DHAR for which we have constructed a plant expression vector. This is the first report of the DHAR cDNA from fruit Tree. It would be helpful about ransferring this gene. So that we can get more information about the role DHAR plays in AsA-GSH cycle and its metabolism pathway. Main results of this study are as follows:1. Three methods were adopted to extract RNA from apple leaves, the results showed that SDS-Hot Hydroxybenzene method was better than others.2. Based on the submitted sequences of DHAR from other plants, we got a fragment of DHAR from apple, then we got the full length of DHAR cDNA by using 3'-RACE and 5'–RACE . The largest ORF codes 314 amino acid. DHAR gene from apple exhibited 76℅,72℅,76℅/69℅ identity respectively with that from tomato, rice and tobacoo(1/2).3. The plant expression vector pSB-DHAR was constructed.