綠竹Bambusa oldhamii蔗糖轉運蛋白質之功能探討

Abstract

高等植物中，蔗糖轉運蛋白質是負責跨膜的蔗糖運輸，它對植物的生長非常重要。先前的研究已從綠竹的cDNA庫中選殖出一段可能為蔗糖轉運蛋白質的cDNA序列 (BoSUT1)，其與植物SUT5類蔗糖轉運蛋白質具高同源性；此段序列已被轉形入酵母菌表現系統 (P. pastoris X-33 and S. cerevisiae SUSY7) 進行重組蛋白質表現，以進行功能性分析。本論文的主要目標是在闡明此蛋白質是否具有蔗糖轉運的功能，並期建立適當的生物物理方法來觀察經由BoSUT1發生的運輸過程。首先，本研究中對BoSUT1核苷酸序列進行修改，以期提高重組蛋白質在酵母菌中的表現量，進而提升功能測定時之訊號強度。其次，以爪蟾卵母細胞表現BoSUT1重組蛋白質，利用蔗糖轉運蛋白質可以同時轉運蔗糖以及質子之特性，追蹤是否有伴隨蔗糖轉移一同發生的質子流。目前為止，本研究在不同的表現系統中皆可成功表現出BoSUT1重組蛋白質，但在功能性測試中分別面臨不同問題，因此尚無法由實驗結果判斷BoSUT1是否具蔗糖轉運之功能。雖然如此，本研究提供了對BoSUT1更多的了解並發現新問題，也為未來的其它研究方法做了更多的準備。

In higher plants, sucrose transporters are responsible for sucrose transportation across membranes and are crucial for plant growth. Previously, a putative sucrose transporter (BoSUT1) with homology to SUT5-type sucrose transporters was cloned from a bamboo (Bambusa oldhamii) shoot cDNA library and transformed into yeast strains (P. pastoris X-33 and S. cerevisiae SUSY7) for expression of the recombinant proteins. The major objectives of this study are to elucidate the function of BoSUT1 and to develop proper biophysical methods for monitoring the transport processes mediated by BoSUT1. First, attempts had been made to enhance the level of recombinant BoSUT1 proteins expressed in yeast by modifying the nucleotide sequence of BoSUT1. Second, Xenopus laevis oocytes were used as the second heterologous expression host to measure the proton flux accompanying sucrose transport activity. By far, the recombinant BoSUT1 proteins have been expressed in both heterologous expression systems but the sucrose transport activity of BoSUT1 has not been detected. However, this work provides more understanding of the problems involved and opens up the possibility for more advanced investigations.