| Summary: | 碩士 === 國立嘉義大學 === 農藝學系研究所 === 99 === Aldose reductase (AR) belongs to AKR (aldo-keto reductase) superfamily. AR is NADPH-dependent and widely distributed in animals and plants. It can catalyze D-glucose converted to D-sorbitol. In previous study, we cloned 5 OsAR genes and observed that OsAR5 was specific expressed on matured grain and root tissue. Besides, it could be enhanced by ABA and osmotic stress treatment. In our study, we set up the OsAR5 over-expression lines (OxAR5s), T-DNA knockout lines (M16610) and wild type (TNG67). At present study, we analyzed the enzyme activity and protein levels (by western blot) to clarify these three types of rice. The phenotypic observation and chlorophyll fluorescence (Fv/Fm) determination from seedling were used as the indicator for rice under high temperature and chilling stress. The results showed OxAR5s transformants maintain higher Fv/Fm than M16610 and TNG67 either under 43℃ or 4℃ treatment. Besides, we also found that OxAR5s transformants reduced H2O2 and O2- content under both temperature stresses by 3,3’- diaminobenzidine and nitroblue tetrazolium staining. These results suggested that OsAR5 could be related with temperature stress tolerance by reducing H2O2 and O2- content.We cloned OsAR5 promoter at upstream 1041 bp from start codon, then fused with GUS (β-glucuronidase) reporter gene. The plasmid was transformed into TNG67 (Oryza sativa L. cv. TNG67) by Agrobacterium-mediated method. Totally, 9 of transgenic cell lines (TCLs) were amplified on selection medium and 10 T0 transformants were further regenerated. The GUS staining is enhanced by temperature stress (4℃, 43℃ ) from TCL6, 8, and 9. In addition, the TCL1, 5, 6, and 7 showed GUS positive stained under salt or osmotic stress treatment. The GUS staining is enhanced from leaves and roots of T0 transformants by variable abiotic stresses similar to the result from TCLs. These results indicated that OsAR5 could be induced by temperature, osmotic and oxidative stress. All the result indicated that OsAR5 involved rice temperature and osmotic stress regulatory.
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