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研究生: 田詩婷
Tasman, Marcelline
論文名稱: 分解葉片老化過程中參與類囊膜瓦解蛋白
Identification of Putative Proteins in Thylakoid Membrane Degradation during Leaf Senescence
指導教授: 李瑞花
Lee, Ruey-Hua
共同指導教授: 黃浩仁
Huang, Hao-Jen
學位類別: 碩士
Master
系所名稱: 生物科學與科技學院 - 生命科學系
Department of Life Sciences
論文出版年: 2013
畢業學年度: 101
語文別: 英文
論文頁數: 116
外文關鍵詞: Arabidopsis, chloroplast, glycosylhydrolase, hydrolysis, leaf senescence, lipases, polar acryl lipids, thylakoid membrane
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  • Chloroplasts are photosynthetic green plastids characterized by the intricate three-dimensional structure of thylakoid networks in higher plant. Disassembly of the thylakoid membranes is the first step leading to chloroplast degradation, nevertheless the degradation processes involved is not understood. Chloroplast membranes contain four major polar acryl lipids including uncharged galactolipids digalactosyldiacylglycerol (DGDG) and monogalactosyldiacylglycerol (MGDG), and negatively charged phospholipids mainly phosphatidylglycerol (PtdPG) and sulpholipid, sulphoquinovosyldiacylglycerol (SQDQ). Two function groups of enzymes, glycosyl hydrolases and lipases, are involved in the hydrolysis of these lipids based on chemical structures of these polar lipids. We used Arabidopsis as model plant. In this study, we analyzed the expression patterns of all the putative glycosyl hydrolases capable of hydrolyze the thylakoid membranes including 6 α-galactosidases (EC 3.2.1.22), 18 β-galactosidases (EC 3.2.1.23) and 2 α-D-glucosidases (EC 3.2.1.20) during different stages of leaf development and senescence. We also have retrieved 2500 nuclear genes encoding chloroplast protein in the Arabidopsis genome. ~ 100 of these proteins were selected for further analysis aim to identify proteins responsible for initial degradation of thylakoid membranes inside the chloroplast.The expression patterns of these genes analyzed by using RT-PCR during leaf growth and senescence were investigated using rosette leaves after 6 – 16 week of sowing were presented and discussed.

    CONTENTS Abstract ………………………………………………...………………………………………i Acknowledgement ……………………………………….……………………………………ii Contents……………………………………………………………………………………….iii List of Figures ……………………………………………………………………………..…vi List of Tables ………………………………………………………………………………...vii Abbreviations………………………………………………………………………………...viii Chapter 1 Introduction ………………………………………………………………………...1 1.1. Leaf senescence and regulation…………………..………………………………….1 1.1.1. Definition of leaf senescence…………..…………………………………1 1.1.2. Regulation………………………………..……………………………….1 1.1.3. Degradation process during leaf senescence...……………………………2 1.1.3.1.Chlorophyll degradation …………………...…………………………..3 1.1.3.2. Protein degradation……….…………………………….……………..4 1.1.3.3. Lipid degradation………….…………………………………………..5 1.1.3.4. Nutrient recycling ……………………………………………………. 6 1.2. Biosynthesis of thylakoid membrane lipids and theirstructural function in chloroplast……………………………….…………………………………………….7 1.2.1. Monogalactosyldiacylglycerol (MGDG) .……………………………………..9 1.2.2. Digalactosyldiacylglycerol (DGDG)…………………………………………10 1.2.3. Sulfoquinovosyldiacylglycerol (SQDG) and Phospatidylglycerol PG)……...11 1.3. Degradation of thylakoid membrane during leaf senescence ……………………….11 1.3.1. Galactolipid degradation…………………………….………………………..13 1.3.2. Phospolipid degradation ……………………………………………………...14 1.3.3. SQDG degradation………………………………….…………………….…..14 1.4. Glycosyl hydrolase and lipase related to thylakoid membrane degradation………...15 1.4.1. -galactosidase………………………………………………………………..16 1.4.2.-galactosidase…….…………………………………………………………..17 1.4.3.-glucosidase……………………………………………………………….....17 1.4.4.-glucosidase…………………………………………………………….……17 1.5. Purpose………………………………………………………………………….…....18 1.5.1. Research aim…………………………………………………………….……18 1.5.2. The proposed works…………………………………………………….…….19 Chapter 2 Materials and methods…………………………………………………….….……20 2.1. DNA sequences from database………………………………………………….….20 2.2. Sequence alignment and analysis ……………………………………………….….20 2.3 Plant Material…………………………………………………………………….….20 2.4. Preparation of total RNA…………………………………………………………...20 2.5. Semi-quantitative reverse transcription PCR (RT-PCR)…………………………...21 2.6. Polymerase Chain Reaction (PCR)…………………………………………………21 2.7. DNA electrophoresis analysis………………………………………………………22 2.8. Total protein extraction and content……………………………………….…..…...22 2.9. Western blot analysis ………………………………………………………………22 2.10. Total chlorophyll extraction and determination…………………………………..23 Chapter 3 Results……………………………………………………………………………..24 3.1. Hydrolytic enzyme cleavage sites for thylakoid membrane lipids degradation by glycosyl hydrolases and lipases…………………………………………………….24 3.2. The phylogenetic analysis of putative glycosyl hydrolase in thylakoid membrane Degradation………………………………………………………………………….24 3.2.1. Acidic -galactosidase ……………………………………………………….25 3.2.2. Alkaline -galactosidase……………………………………………………..25 3.2.3. - galactosidases……...………………………………………………………25 3.2.4. -glucosidases ………….…………………………………………………….26 3.2.5. -glucosidases ………….…………………………………………………….26 3.3. Different Stages of leaf development and senescence…………………………………...26 3.3.1. Protein and chlorophyll levels change during different stages of leaf development…………………………………………………………………. 26 3.3.2. Expression pattern of putative proteins prediction …………………………..27 Chapter 4Discussion…………………………………….…………………………………....31 4.1. Growth and biochemical changes during senescence ……………….………...31 4.2. Expression patterns of glycosyl hydrolases and putative chloroplast proteins in thylakoid membrane degradation and catabolism…………………………….32 Chapter 5Conclusion…..………………………………………………………………….….39 Chapter 6 References………………………………………………….…………….………..40 Appendices Appendix 1a-t : Gene expression patterns for putative genes involved in thylakoid membrane degradation after 6-16 weeks of planting.……..........93 Appendix 1u.:Gene expression patterns for internal control genes, Ubiquitin-10 (UBQ10), SAG-12, and Rubisco activase, in the rosette leaves after 6-16 weeks of planting……………………………………………………..113 Appendix 2 : Statistic analysis results………………………………………………114

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