Cloning And Functional Analysis Of Dehydroascorbate Reductase Gene In Potato

Ascorbic acid (L-ascorbic acid, AsA) is a major antioxidant for plants and human. In contrast to most animals, humans lack the ability to synthesize AsA, it must be obtained from food regularly. Hence, it has became one of the most important nutritional quality factors in many horticultural crops. In plant, AsA is an important metabolites that serves many functions such as protecting plants against oxidative damage, photoprotection, controling plant growth and so on.Dehydroascorbate reductase (DHAR; EC 1.8.5.1) is the key enzyme of the ascorbate-glutathione cycle (AsA -GSH cycle). In this study, we isolated and characterized the cDNAs encoding two isoform DHARs localized in cytosol and chloroplasts respectively from potato. Both the relationship between DHAR genes expression and AsA organs distribution in different organs and the relationship between the change of DHAR genes expression and the tolerance to temperature stress in potato were investigated. For Further analysis of DHAR physiological function, we developed transgenic potato plants that overexpress the two genes respectively. The main results are as follows:1. AsA was accumulated in young leaves and tuber. AsA pool size (the sum of AsA and DHA) in leaves and stems was correlated with L-galactono-1,4-lactone dehydrogenase (GalLDH) activity. AsA content was significantly related with the DHAR activity in all organs, especially in tubers. This suggested that high AsA content in young leaves might result from higher activites of GalLDH and DHAR, while AsA accumulation in tuber might be attributed to the transportation from leaves, and DHA recycling via DHAR.2. In order to investigate the effects of high and low temperature stresses on AsA metabolism system in potato leaves, potato plants were exposed to high (40℃) or low (5℃) temperature stress respectively. The results showed that the AsA content increased in the early treatment of 40℃and 5℃, then followed by a continuous decline. The activities of DHAR, GalLDH, APX, MDHAR and GR increased in the early treatment time and decreased after it reached the highest point. The transcripts level of DHAR genes showed the same changes as the related enzyme activities. It suggested that the expression of two DHAR genes were induced to regulated the AsA, and thus to protect plants against oxidative damage imposed by temperature stresses.3. The two cDNA encoding these DHARs were successfully isolated from potato leaves, designated as SrDHAR1 and SrDHAR2, and their nucleotide sequences had been submitted to GenBank databases under accession numbers EF030707 and FJ477252. The analysis based on the deduced amino acid sequence of SrDHAR1 and SrDHAR2 demonstrates that the proteins encoding by two genes might have the same function, the only difference is SrDHAR2 has a chloroplast transit peptides (cTP) that directs it to the chloroplast stroma after the biosynthesis of protein compeleted.4. The two full-length cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense constructs respectively. And then the constructs were introduced into potato and verified by PCR and Real time PCR. The result indicated that the SrDHAR1 and SrDHAR2 had been recombined into potato genome and both transgenic potato plants were obtained.5. The over-expression of SrDHAR1 significantly increased DHAR activity, AsA content, AsA+DHA content and AsA/DHA in both leaves and tubers. While, the increase in SrDHAR2 expression increased DHAR activity, the contents of AsA and AsA+DHA ,and AsA/DHA in leaves, but these values in tubers did not show significant change which suggested that its over-expression did not affect the tubers DHAR activity and AsA content.6. The over-expression of SrDHAR1 and SrDHAR2 significantly increased chlorophyll content and photosynthetic rate of leaves, especially in the plants of overexpressing SrDHAR2. Therefore, the overexpression of DHAR in chloroplast showed more significant effects on the chlorophyll protection and photosynthesis increase than the over expression in the cytosol.7. The transgenic potato showed stronger growth than wild type plant. The over-expression of SrDHAR1 and SrDHAR2 increased plant height and the leaf area, moreover the increase in SrDHAR1 expression increased stem diameter. It suggested that over-expression of SrDHAR1 and SrDHAR2 could promote the growth of potato plant in a certain degree.