赤黴素有多種生物學功能，包括促進莖的伸長、種子萌發以及花的發育。玉米赤黴素缺陷型突變體dwarf1 (d1) 表現出植株矮壯和雌雄兩性花，即原為雌花部位發育出雙性花。但是該突變的分子基礎尚不清楚。通過分析多個d1等位基因突變體的分子組成特征，我們證明d1突變體是由能催化赤黴素中間代謝物轉變為活性赤黴素的赤黴素3-氧化酶（ZmGA3ox2） 突變引起的。重組D1 蛋白能於體外催化至少4個反應，包括GA20 轉變為GA3，GA5轉變為GA3，GA20轉變為GA1 以及GA9轉變為GA4等。煙草細胞中D1-GFP 的瞬時表達和細胞組分蛋白質印染分析等兩個獨立的方法，揭示了D1 蛋白是雙定位於細胞核和細胞質中。此結果暗示活性赤黴素能夠在此兩種細胞器中合成。這個雙定位的結果與赤黴素受體GID1蛋白的定位壹致。在早期的玉米雌花發育的過程中，D1 蛋白特異且大量地表達在雌花中的雄花原基細胞，揭示了赤黴素發揮其抑制雄花原基發育的功能需要在該組織大量合成。 / DELLA 蛋白是赤黴素信號傳導的壹個阻遏物。玉米包含僅壹個DELLA蛋白命為DWARF8（D8）。發生在D8 蛋白N端的突變產生了赤黴素非敏感型突變體dwarf8 （d8）。d8突變體與d1突變體有很多共同特征，包括侏儒植株，深綠色的葉片以及雌雄兩性花。這些特點都表明玉米對赤黴素的響應是被DELLA蛋白所抑制的。DELLA蛋白是通過蛋白互作的方式來限制其互作蛋白功能，以達到抑制赤黴素下遊信號傳導的目的。多樣的赤黴素響應必然需要多樣的DELLA互作蛋白。基於赤黴素在調控玉米性別決定過程當中的獨特功能，我們提出假設：玉米當中存在未知的D8互作蛋白。通過酵母雙雜交方法篩選玉米雌花的cDNA文庫，找到14個在酵母系統與D8互作的蛋白。ZmSPX1是這些蛋白中的壹個但不清楚功能。通過雙分子熒光互補實驗和蛋白質體外結合實驗，D8和ZmSPX1之間的互作被進壹步確定。基于此，我们找到数个候选的D8互作蛋白以及证明了ZmSPX1是D8真正的互作蛋白；这些工作都为进一步研究ZmSPX1蛋白在调控性别分化、细胞分裂和分化等赤霉素响应中的作用提供了基础。 / Gibberellins (GA) have multiple biological functions including promoting stem elongation, seed germination and flower development. The GA deficient dwarf1 (d1) mutant in maize displays plant dwarfism and andromonoecy, i.e. forming anthers in the female flower. However, the molecular basis is not clear. Through molecular characterization of multiple d1 alleles, I prove that the d1 is caused by mutations in the GA 3-oxidase (ZmGA3ox2) that converts the inactive GA intermediates to bioactive GAs. The recombinant D1 protein catalyzes at least four reactions in vitro, converting GA20 to GA3, GA5 to GA3, GA20 to GA1 and GA9 to GA4. Subcellular localization analysis by two independent approaches which are in vivo D1-GFP analysis and western blot analysis of organelle fractions revealed that the D1 protein is dual-localized in the nucleus and the cytosol. ZmGA20ox was also localized in both the cytosol and the nucleus by the in vivo GFP fusion analysis. Interestingly, the dual-localization of D1 and ZmGA20ox coincides with the localization of the GA receptor GID1. In early phase of maize female flower development, the D1 protein was found specifically and highly expressed in the stamen primordia within the female florets. These results indicate that bioactive GAs can be synthesized in both the cytosol and the nucleus, and that the suppression of stamen in female florets is mediated by locally synthesized GAs. This finding provides new insights to the understanding of GA biosynthesis and signal transduction in plants. / DELLA proteins are repressors of GA signal transduction. DWARF8 (D8) is a DELLA protein in maize, Mutations in the N-terminal of D8 resulted in dominant GA insensitive dwarf8 (d8) phenotype. d8 displayed similar phenotypes as the d1mutants; i.e. plant dwarfism, dark green leaves and andromonoecy, indicating that D8 is a master repressor mediating these GA functions. DELLA proteins suppressed the downstream signal transduction of GA by restricting their interacting protein functions through protein-protein interaction. Diverse GA responses require numerous DELLA interacting proteins. Based on the unique function of GA in regulating sex determination in maize, I hypothesize that D8 mediates the GA responses by interacting with yet unknown proteins in maize. Through yeast two hybrid screening of the maize ear cDNA library, I identified 14 proteins that showed genuine interaction in yeast system. Among these, a SPX domain containing protein named as ZmSPX1 was present. SPX domain containing proteins in yeast are implicated in cell cycle regulation; however, their functions in plants are unknown. GFP fusion analysis indicated that ZmSPX1 co-localizes with D8 in the nucleus and their interaction was confirmed by bimolecular fluorescence complementation (BiFC) and in vitro pull-down assay. To this point, I have identified several candidates for D8 interacting proteins and provided strong evidence that ZmSPX1 is a bona fide D8 interacting protein which set a foundation for further analysis of its function in mediating GA responses including sex determination, cell division and elongation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Yi. / Thesis (Ph.D.) Chinese University of Hong Kong, 2015. / Includes bibliographical references (leaves 60-67). / Abstracts also in Chinese. / Chen, Yi.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1077650 |
| Date | January 2015 |
| Contributors | Chen, Yi , active 2015 (author.), Zhang, Jianhua , 1956- (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Life Sciences, (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource], electronic resource, remote, 1 online resource (xvi, 68 leaves) : illustrations (some color), computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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