| Summary: | 碩士 === 國立嘉義大學 === 農藝學系研究所 === 100 === It has been known that a rice cysteine proreinase, OsEP3A（Oryza sativa endoproteinase 3A）, gene expression was up-regulated during germinating seeds or cells were grown on nitrogen free（-N） medium. In the present study, serial deletion and internal sequence deletion of the OsEP3A up-stream regulatory sequences（URS） were fused with luciferase（LUC） or β-gluronidase（GUS） as a reporter. These expression vectors were transferred into rice calli by Agrobacterium-mediated gene transformation. The transformed calli and transgenic seeds were selected randomly and cultured in +N or –N of MS medium for 5 days, respectively, and LUC and GUS activities were analyzed. We found that calli which were transformed with 837::GUS, 531::GUS（LUC）, 361::GUS, D236/183::GUS（LUC） and D183/113::GUS（LUC）, the LUC activities and the depth of GUS coloration were higher, when cells were cultured in –N medium compared with in +N medium. On the contrary, the transformed calli of 236::GUS（LUC）, 183::GUS（LUC）, 113::GUS（LUC） and D278/236::GUS（LUC）, or 35mP::GUS（LUC）, there was no significant difference in LUC activity or coloration of GUS staining , whether cells were cultured in + N or -N medium. These results suggest that the nitrogen responsive sequence（NRS） was located from -278 to -236 of the OsEP3A promoter. In order to identify the GA- and submergence-responsive sequence in OsEP3A URS, transgenic seeds were germinated for 1-5 d treated with three various conditions, containing normal, flooding, or flooding plus ethylene biosynthesis inhibitor CoCl2. The coloration of GUS were found in germinating seeds of 837::GUS, 531::GUS, 361::GUS, D236/183::GUS and D183/113::GUS, but did not found in 236::GUS, 183::GUS, 113::GUS, D278/236::GUS and 35mP::GUS. Based on these results, suggesting that the OsEP3A gene expression was regulated by GA, whether rice seeds were germinated either in normal or in flooding conditions. Moreover, the GA responsive sequence might be located donstream of -361 of OsEP3A promoter, and at least one cis-element for GA responses which resides in -278- -236 region.
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