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研究生:石伊雯
研究生(外文):Shih Yi-Wen
論文名稱:D-甘露糖導致水稻細胞死亡過程中酪胺酸磷酸化之研究
論文名稱(外文):Tyrosine - Specific Protein Phosphorylation During D-mannose Induced Cell Death in Rice Cells
指導教授:黃浩仁劉 景 煌
指導教授(外文):Hao-Jen HuangZin-Huang Liu
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:52
中文關鍵詞:訊息傳遞路徑酪胺酸磷酸化訊程式性細胞死亡
外文關鍵詞:Programmed cell deathSignalling transduction pathwaysTyrosine phosphorylation
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中 文 摘 要
蛋白質酪胺酸磷酸化在動物細胞中已有廣泛的研究且已知在動物細胞的程式性死亡中扮演一個重要的角色。然而, 酪胺酸磷酸化的現象在植物細胞的程式性死亡過程中仍屬未知。此實驗證實了D-甘露醣在水稻的懸浮細胞中會引起水稻懸浮細胞的死亡以及DNA的斷裂。水稻懸浮細胞以D-甘露糖處理24小時後, 兩個蛋白質分子20 kDa與43 kDa酪胺酸磷酸化的現象明顯上升。水稻懸浮細胞在D-甘露糖中經過3天的處理後, 兩個蛋白質分子16 kDa與20 kDa酪胺酸磷酸化的現象明顯降低。此外, 在老化的水稻懸浮細胞中也發現有DNA斷裂的現象以及兩個蛋白質分子26kDa與40kDa酪胺酸磷酸化的現象。同時, 在D-甘露糖處理過程中也發現兩個MAPKK (mitogen-activated rotein kinase kinase) (MEK) /MAPK訊息傳導途徑的基因OsMEK以及 OsMAPK2 的表現量明顯增加。本實驗證明酪胺酸磷酸化改變的現象以及(MEK)/MAPK訊息的傳導和水稻細胞的死亡有關。
ABSTRCT
In mammals protein tyrosine phosphorylation plays an important role in the activation of programmed cell death. However, tyrosine phosphorylation involved in cell death has not been examined in plants. These studies demonstrated that D-mannose induced cell death and DNA fragmentation in rice suspension cells. In the presence of mannose for 24 hours, tyrosine phosphorylation of two proteins, 20 kDa and 43 kDa markedly increased. After incubating 3 days, the level of phosphotyrosine accumulation declined in bands of 16 and 20 kDa. In addition, the occurrence of DNA fragmentation and two tyrosine-phosphorylated proteins, 26 kDa and 40 kDa, were detected in aged suspension-cultured cells. The expression of genes that encode mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK signalling pathway, OsMEK and OsMAPK2, are up regulated during D-mannose treatment. The results provide evidence that protein tyrosine phosphorylation as well as MEK/MAPK signalling pathway is associated with cell death in rice.
CONTENTS
ACKNOWLEDGEMENTS…………………………………………………….…….1
CHINESE ABSTRACT………………………………………………………………2
ABSTRACT…………………………………………………………………………..3
CONTENTS……………………………………………………………………..……4
LIST OF TABLE………………………………………………….….……………….6
LIST OF FIGURES………………………………………………………….….…….7
ABBREVIATION……………………………………………………………….…....9
I. INTRODUCTION…………………………………………………………………10
1. Programmed cell death……………………………………………………..….11
1.1 Morphological characteristics of programmed cell death…………………..11
1.2Biochemical hallmark of programmed cell death…………………………12
2. Programmed cell death in plants……………………………………………….13
3. Cytochrome C mediates PCD………………………………………………….14
3.1 Cytochrome c induce caspase activation…………………………………..14
3.2 Involvement of caspase-like proteases in plants……………………………15
4. Tyrosine phosphorylation in programmed cell death…………………………...15
4.1 Plant protein kinase and signal transduction……………………………….15
4.2 Protein tyrosine phosphorylation regulates apoptosis………………………16
5. The MEK/MAPK pathway in the regulation of PCD…………………………..17
5.1 The MAP kinase pathways………………………………………………...17
5.2 Involvement of a MAPK signalling pathways in apoptosis………….……18
6. The effect of D-mannose on plant metabolism………………………………..19
7. The purpose of this study………………………………………………………20
Ⅱ. MATERIALS AND METHODS…………………………………………….……21
1. Plant Material and Treatment…………………………………………………...21
2. Viability Staining………………………………………………………………..21
3. Fresh Weight and Dry Weight Determination……………………………….….21
4. TUNEL Assay…………………………………………………………………..21
5. DNA Ladder Analysis…………………………………………………………..22
6. Preparation of Protein Extracts………………………………………………….23
7. Electrophoresis and Western Blot Analysis…………………………………23
Ⅲ.RESULTS……………………………………………….25
1. D-mannose induces cell death in rice cells……………………………………..25
2. Detection of DNA fragmentation in D-mannose-treated rice cells…….………25
3. Changes in protein tyrosine phosphorylation during D-mannose
induced cell death……………………………………………………………….26
4. DNA fragmentation is associated with aging…………………………………..27
5. Tyrosine phosphorylation of two proteins, 26kDa and 40kDa, occurs in
aged rice suspension-cultured rice cells…………………………………………27
Ⅳ.DISCUSSION…………………………………………….28
Ⅴ. REFERENCES…………………………………………………32
Ⅵ. APPENDIX
Involvement of a MAPK signalling pathway in D-mannose treated
cultures……………………………………………………………………………….50
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