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研究生:呂昂樹
研究生(外文):Ong-Suh Lu
論文名稱:綠竹Bambusa oldhamii蔗糖轉運蛋白質之功能探討
論文名稱(外文):Studies on the function of sucrose transporter BoSUT1 in Bambusa oldhamii
指導教授:梁國淦
指導教授(外文):Kuo-Kan Liang
口試委員:楊啓伸蔡宜芳周百里
口試委員(外文):Chii-Shen YangYi-Fang Tsay
口試日期:2013-05-09
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:71
中文關鍵詞:綠竹蔗糖轉運子
外文關鍵詞:bamboosucrose transporter
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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.




Contents i
Abbreviation List v
Abstract vi
中文摘要 vii
Chapter 1. Introduction 1
1.1 Sucrose in higher plants 1
1.2 Sucrose transport in plants 1
1.3 Sucrose transporters (SUTs) 2
1.3.1 Subfamily SUT1 3
1.3.2 Subfamily SUT2 4
1.3.3 Subfamily SUT3 4
1.3.4 Subfamily SUT4 5
1.3.5 Subfamily SUT5 5
1.4 Lipid rafts and SUT 6
1.5 Sucrose transporters in Bambusa oldhamii 7
Chapter 2. Materials and Methods 9
2.1 Construction of expression plasmids 9
2.1.1 Plasmids for heterologous expression in yeast 9
2.1.2 Plasmids for heterologous expression in oocytes 14
2.2 Heterologous expression 15
2.2.1 Expression in yeast 16
2.2.2 Expression in Xenopus Oocyte 17
2.3 Extraction of membrane proteins 19
2.3.1 Isolation of yeast membrane fraction 19
2.3.2 Isolation of the detergent resistant membrane fraction of yeast 19
2.3.3 Isolation of oocyte membrane proteins 20
2.4 Complementation functional assay 21
2.5 Sucrose uptake assay 21
2.6 Functional assay in Xenopus oocytes 22
Chapter 3. Results and discussion 24
3.1 Characterization of the recombinant BoSUT1 proteins expressed in yeast 24
3.1.1 Expression of the recombinant sucrose transporter proteins in yeast 25
3.1.2 Complementation functional assay 26
3.1.3 Sucrose uptake assay 27
3.1.4 Detection of sBoSUT1 in membrane fraction and DRM 28
3.2 Functional assay in Xenopus oocytes 29
3.3 Discussion 33
Chapter 4. Perspectives 37
4.1 Improvement of the present study 37
4.1.1 Enhancement of protein expression 37
4.1.2 Confirmation of yeast strain 37
4.1.3 Detection of intracellular pH 38
4.1.4 Detection of extracellular pH 38
4.2 Further experiments 39
4.2.1 Plant hosts 39
4.2.2 Spectroscopic analysis 40
References 43
Figures 52
Tables 63
Appendix 66


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