| Summary: | 碩士 === 國立臺灣大學 === 農藝學研究所 === 100 === In this study, anther-specific promoters were cloned from rice which highly express in rice anther. In order to characterize the functions of promoters, the reporter gene (gus and gfp) was constructed. Then, a lethal system was established with lethal gene barnase to obtain male sterile transgenic plants.This study comprises three parts. Previously, high TPM (transcripts per million) genes of seven rice anther development stages were selected by Huang. Based on NCBI rice genomic DNA database, specific primers were designed to clone 1.5Kb putative DNA fragment of anther-specific promoters by PCR, which were named P13 and P41. Second, the 35S promoters in pCAMBIA1301 and pCAMBIA1303 plasmids were replaced by P13 and P41 promoter to construct reporter gene system, C1301-P13, C1303-P13, C1301-P41 and C1303-41. Third, the lethal system, P13bar, was achieved by inserting P13 promoter flanking the barnase gene.The resulted five constructs were introduced into rice, tobacco and Arabidopsis plants by Agrobacterium-mediated transformation system. Reporter gene containing transgenic rice, tobacco and Arabidopsis plants were subjected to GUS expression assays. GUS staining results revealed that P13 promoter expressed GUS in microspores of rice and anther, sepals and stigma of tobacco. P41 promoter expressed GUS in tapetum and microspores of rice. Furthermore, GUS activity was not dected in flowers but in seeding of Arabidopsis Pollen activity of lethal system transgenic Arabidopsis was confirmed by TTC staining. Transgenic Arabidopsis pollens containing P13bar showed no lethal effect. In the future, expression stage and tissue of P13 and P41 promoters will be further characterized and P41bar lethal system will be constructed. This two anther-specific promoters many be applied in dual inducible system for hybrid rice experiments.
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