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標題: | 綠竹 Bambusa oldhamii 之蔗糖轉運:蔗糖轉運子與蔗糖轉化酶之功能探討 Sucrose transport in green bamboo Bambusa oldhamii:Studies on function of sucrose transporter and cell wall invertase |
作者: | Shu-Chien Liao 廖述建 |
指導教授: | 王愛玉 |
關鍵字: | 蔗糖轉化酶,蔗糖轉運子,脂筏體,免疫共沉澱,啟動子, invertase,sucrose transporter,lipid raft,Co-immunoprecipitation,promoter, |
出版年 : | 2013 |
學位: | 博士 |
摘要: | 摘要
在高等植物中,蔗糖轉運子與細胞壁蔗糖轉化酶在蔗糖轉運過程中扮演重要的角色,蔗糖轉運子負責將蔗糖以跨越膜系方式進行轉運,而細胞壁蔗糖轉化酶是將胞外蔗糖水解成葡萄糖及果糖,再經由六碳糖轉運子運至細胞中,本論文首要目的是探討這兩個蛋白質在綠竹中之功能。先前研究已從綠竹筍cDNA 庫中選殖出一段蔗糖轉運子之cDNA 序列 (BoSUT1),本研究利用酵母菌表現系統表現重組 BoSUT1 蛋白質,然而,活性及功能性互補試驗結果顯示,在酵母菌中表現之重組 BoSUT1 蛋白質不具蔗糖轉運之活性。細胞內定位分析顯示,BoSUT1 不均勻分布在原生質膜上,可能存在脂筏體中。免疫共沉澱分析發現 BoSUT1 可能與其他蛋白質有交互作用。此外,本研究也選殖了 BoSUT1 之啟動子序列,並找到一些可能參與基因調控之順式調控因子。進一步以培養於試管之綠竹苗作為材料,以 real-time PCR 法來探討不同因子對 BoSUT1 基因表現的影響,結果顯示, BoSUT1 mRNA 量會受到 indole-3-acetic acid、benzylaminopurine 及蔗糖消耗而增加。除了 BoSUT1 外,我們也選殖三個蔗糖轉化酶 (Boβfruct1、Boβfruct2 及 Boβfruct3) 基因之啟動子序列,並分析此三個基因表現情況。由 real-time PCR 分析結果,推測此三個 Boβfruct 基因各自在植物適應環境變化過程中扮演獨特的角色。此外,Boβfruct2 對於細胞外之蔗糖可立即作用,並且對於積貯活性 (sink activity) 的維持扮演必要的角色;Boβfruct3可能為光訊息傳導及 ABA 訊息傳導路徑的一個交叉點。分析未出土綠竹筍粗抽液及部份純化之酵素分劃中發現了較高分子量之蔗糖轉化酶,推測蔗糖轉化酶除了在轉錄層次上受到調控外,也可能具有轉譯後修飾。P. pastoris 表現並純化之重組 Boβfruct3 蛋白質可以在試管中被 SUMOylation;然而,這種修飾作用是否會在植物體內中發生仍不清楚。綜合上述研究結果,顯示BoSUT1 及三種酸性蔗糖轉化酶在綠竹中會受到高度調控,證實這些蛋白質在綠竹生長發育、代謝及逆境反應過程之重要性。 Abstract In higher plants, sucrose transporters and cell wall invertases play crucial roles in sucrose transportation. Sucrose transporters are responsible for sucrose transport across the membrane system; cell wall invertases catalyze the hydrolysis of apoplastic sucrose to glucose and fructose. The hexoses are in turn transported into cells via hexose transporters. The major objective of this study is to elucidate the roles of the two proteins in bamboo. A putative sucrose transporter (BoSUT1), which was cloned from a bamboo shoot cDNA library and belonged to the SUT5-type sucrose transporters, was transformed into yeast strains for expressing the recombinant proteins. The results of sucrose uptake assay and complementation assay indicated that the BoSUT1 expressed in yeast could not transport sucrose into cells and also failed to complement yeast mutant lacking sucrose transport activity. Subcellular localization analysis showed that BoSUT1 was distributed heterogeneously in the plasma membrane and predominantly present in lipid raft-like structures. Co-immunoprecipitation assay showed that BoSUT1 might interact with other proteins. The promoter regions of BoSUT1 were cloned, and putative regulatory cis-elements were identified. The expression of BoSUT1 in multiple shoots of bamboo that were cultured in vitro under different conditions was analyzed by real-time PCR. The levels of BoSUT1 mRNA were increased by indole-3-acetic acid, benzylaminopurine and the depletion of sucrose. In addition to BoSUT1, the promoter regions of three acid invertase genes (Boβfruct1, Boβfruct2 and Boβfruct3) of bamboo were also cloned and their expression patterns were analyzed. The results of real-time PCR suggested that these three Boβfruct genes have individual roles in the adaption of the plant to environmental changes. Boβfruct2 might also have an essential role in the immediate response of cells to sucrose availability and in the maintenance of sink activity. Moreover, Boβfruct3 might be one of the interacting nodes of the light and ABA signaling pathways. In addition to being regulated at the transcriptional level, the Boβfruct-encoded enzymes might be posttranslationally modified as revealed from the presence of invertases with molecular weight higher than the predicted ones in the crude extract of etiolated bamboo shoots and the partially purified enzyme fractions. An in vitro SUMOylation assay of the recombinant Boβfruct3 proteins that were expressed and purified from P. pastoris showed that the recombinant proteins were able to be SUMOylated; however, whether this modification occurs in planta remains unknown. Taken together, the results of this study show that the expressions of BoSUT1 and different isoforms of acid invertases in bamboo are under complex regulation, which highlights the importance of these proteins in the growth, development, metabolism and stress responses of this plant. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62043 |
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