請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/52925
標題: 竹筍粒線體能量守恆及能量消散系統之蛋白質體及基因體分析
Proteomic and Genomic Analysis of Bamboo Mitochondrial Energy-Conserving and Energy-Dissipating Systems during Shooting Stage
作者: 簡麗鳳
關鍵字: 基礎研究
bamboo mitochondria
食品科技
竹筍粒線體
能量守恆
能量消散
氧化磷酸化作用
電子傳遞鏈
替代氧化脢
去偶合蛋白
活性氧消除.
蛋白質體
基因體
energy-conversing
energy-dissipating
oxidativephosphorylation
electron transport chain
AOX
UCP
ROS scavenging enzymes
proteome,genome
摘要: 竹筍是亞洲重要經濟作物之一,新筍生長相當快速,推測此時能量需求及轉換效率應很高,若能瞭解其能量代謝機制,有助於經濟作物之發展。粒線體是植物細胞的發電廠,受質在粒線體被氧化時,所產生氫離子或電子的會轉移至電子傳遞鏈,引發許多的氧化還原反應,最後電子被O2 所接受。在電子傳遞過程中,粒線體內外膜會形成質子電化學電位島貢H+。這自由能可以用來推動磷酸化反應,ATP 合成.催化ADP 形成ATP。這種氧化作用和磷酸化作用的偶合,稱之為氧化磷酸化作用,此即粒線體能量守恆系統。但是,粒線體亦存在能量消散系統。例如替代氧化脢(AOX)和去偶合蛋白(UCP),兩者皆能夠抵消氧化磷酸化作用所產生的質子電化學電位(島貢H+)。另外,活性氧(ROS)之產生,亦是能量消散之一種。然而,能量守恆及能量消散系統的調控機制還未被完全瞭解。所以,本計畫欲結合蛋白質體及基因體分析技術,研究竹筍粒線體之能量守恆及能量消散系統的調控機制。預定二年完成,現將預定完成目標分述如下:〈一〉分離竹筍粒線體:先將綠竹筍和冬筍搾汁,再將粗萃取液以梯度法純化粒線體。測定呼吸作用活性,分析氧化磷酸化作用之變化。〈二〉溶解竹筍粒線體蛋白質: 利用非離子性介面活性劑digitonin 、n-dodecylmaltoside、Triton X-100 將粒線體可溶性或膜蛋白完全溶解出來。〈三〉蛋白質體分析技術:(1)以一維凝膠電泳Blue-Native PAGE 或Colorless-NativePAGE 分析綠竹筍和冬筍之粒線體蛋白,可利用酵素活性染色。(2)續以二維凝膠電泳Blue-Native PAGE 或SDS-PAGE 分析,用Coomassie Blue 或銀染。比較分析(a)能量守恆系統之電子傳遞組成及ATP synthase,(b) 能量消散系統之AOX、UCP、ROS 消除.。〈四〉質譜分析:將2-D-PAGE 之樣品以質譜儀(mass spectrometry, MS)之MALDI-TOF及LC-MS-MS 作更詳細的蛋白質身份鑑定。〈五〉建立竹筍粒線體基因體:(1) 先抽取mtRNA,再建立竹筍粒線體cDNA。研究有差異性之complex-I 之mitochondrial-encoded subunits。〈六〉利用生物資訊工具,比對粒線體蛋白質與已知粒線體基因序列的對應關係。
Bamboo is an important economical plant. Bamboo is one of the fastest growing plants onthis planet. It was supposed that the rapid growth requires a huge amount of energysupplement. The understanding of the energy metabolism mechanism will be useful to thedevelopment of economical corps. Mitochondria are the power house of plant cells. Oxidativephosphorylation occurs in plant mitochondria and is the process in which synthesis of ATPfrom ADP and Pi is catalyzed by the H+-ATP synthase as electrons are transferred to O2 byelectron transport chain. Thus, oxidation and phosphorylation are coupled by 島貢H+,composed of a proton gradient (島pH) and an electrical membrane potential (島馬), across theinner mitochondrial membrane; this is the energy-conserving system. In plant mitochondria,contain an alternative there are another free-energy dissipating systems: alternative oxidase(AOX) and uncoupling protein (UCP) that acts as a 島貢H+ dissipating system. In addition, theproduction of reactive oxygen species (ROS) also relate to energy balance. Therefore, the aimof this project is to study the control of energy-conversing and energy-dissipating system inbamboo mitochondria. Proteomic approach will be applied to characterize the mitochondrialfunctional proteins involved in energy metabolism. The project will be focused on:(1) Characterization of bamboo shoot mitochondria: (a) Isolation of bamboo shootmitochondria from summer bamboo B. oldhamii and winter bamboo P. edulis. (b)Evaluate the coupling and uncoupling of oxidative phosphorylation in bamboo shootmitochondria(2 Solubilization of mitochondrial proteins: treatment with nonionic detergents of digitonin,n-dodecylmaltoside, Triton X-100(3) Gel electrophoresis analysis of mitochondrial protein complexes: (a) 1-D electrophoresis:blue-native gel electrophoresis (BN-PAGE) or colorless-native (CN) PAGE will be usedto fractionate mitochondrial protein supercomplexes in their native state. This will beallowed to demonstrate the structure-function relationships of mitochondrial proteins bylocalizing them to specific complexes. (b) 2-D electrophoresis. Then carry out the 2-Delectrophoresis either BN-PAGE/CN-PAGE or SDS-PAGE to identify how thesesupercomplexes interact.(4) Analyze the interested protein using mass spectrometry: MALDI-TOFand LC-MS-MS.(5) Establish of bamboo mitochondrial genome: (a) isolation of total mtRNA; (b)mitochondrial cDNA; (c) isolation of mitochondrial gene which encoding complex Isubunits(6) Bioinformatic analysis: the interested proteins may be further studied using the databaseof mitochondrial proteome and genome.
URI: http://hdl.handle.net/11455/52925
其他識別: NSC96-2313-B005-030-MY2
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1597141&plan_no=NSC96-2313-B005-030-MY2&plan_year=97&projkey=PD9706-0125&target=plan&highStr=*&check=0&pnchDesc=%E7%AB%B9%E7%AD%8D%E7%B2%92%E7%B7%9A%E9%AB%94%E8%83%BD%E9%87%8F%E5%AE%88%E6%81%86%E5%8F%8A%E8%83%BD%E9%87%8F%E6%B6%88%E6%95%A3%E7%B3%BB%E7%B5%B1%E4%B9%8B%E8%9B%8B%E7%99%BD%E8%B3%AA%E9%AB%94%E5%8F%8A%E5%9F%BA%E5%9B%A0%E9%AB%94%E5%88%86%E6%9E%90
顯示於類別:生命科學系所

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