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研究生:沈哲宇
研究生(外文):Che-yu Shen
論文名稱:從甘藷老化葉片分子選殖 mitogen-activated proteinkinase cDNA 及乙烯訊息傳導探討
論文名稱(外文):Molecular cloning of mitogen-activated protein kinase cDNA and study of ethylene signaling in senescent sweet potato leaves
指導教授:陳顯榮陳顯榮引用關係
指導教授(外文):Hsien-jung Chen
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:2011
畢業學年度:99
語文別:中文
論文頁數:96
中文關鍵詞:甘藷乙烯葉片老化有絲分裂活化蛋白質激酶
外文關鍵詞:sweet potatoethyleneleaf senescencemitogen-activated protein kinasePD98059
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乙烯是植物生長調節物質,且在葉片老化的過程扮演一個主要的角色,然而其訊息傳遞的研究在甘藷方面大部份仍然不清楚。Ethephon (釋放乙烯的化合物) 誘導甘藷葉片老化和相關基因的表現受有絲分裂活化蛋白質激酶 (MAPK) kinase inhibitor PD98095的抑制,此結果建議 MAPK cascade 可能參與乙烯誘導甘藷葉片老化和相關基因表現的過程。利用基因專一性引子及 RT-PCR 方法從 ethephon 處理的葉片選殖出一條全長的 cDNA 命名為 SPMAPK,其 open reading frame 總共由1098個核苷酸 (365個胺基酸) 所組成。胺基酸序列比對與植物的 MAPK 具有79.8-83.4 %相似性,且與 Arabidopsis MPK3及 MPK6在親緣樹分析上最為相近。RT-PCR 分析結果顯示 SPMAPK 基因在甘藷的根、莖及葉皆有表現,其中年青成熟的葉片以及部分黃化的老葉表現較多,SPMAPK 的表現亦受 ethephon 誘導而有增強的現象。利用不同的 inhibitors 或 effectors 研究顯示分離的甘藷葉片經 ethephon 處理會加速葉片老化的速度、葉綠素含量降低、光合作用效率 (Fv/Fm 值) 衰減以及誘導相關基因的表現,乙烯受器抑制劑 (1-methylcyclopropene,1-MCP)、MAPK kinase 抑制劑 PD98059、NADPH oxidase 抑制劑 (diphenylene iodonium,DPI)、抗氧化劑 reduced glutathione、Ca2+螯合劑 EGTA 以及新合成蛋白質抑制劑 cycloheximide,都可以延緩或抑制 ethephon 對葉片所造成的效應。根據以上的結果結論,從甘藷葉片分離出一個乙烯可以誘導的有絲分裂活化蛋白質激酶SPMAPK,其在成熟及部分黃化的葉片表現量較多,ethephon 誘導其表現時受1-MCP、PD98059、DPI、reduced glutathione、EGTA 及 cycloheximide 等抑制。此結果亦建議參與在乙烯誘導葉片老化、SPMAPK 及其他相關基因表現可能的訊息因子有乙烯受器,MAPK cascade,增加的過氧化氫,細胞外的鈣離子流入,以及新生合成的蛋白質。一個可能的乙烯訊息傳導模式導致甘藷葉片老化及相關基因表現也被提出討論。
Ethylene is a plant growth regulator and plays a key role in leaf senescence. Its signaling, however, remains mostly unclear in sweet potato. Ethephon, an ethylene releasing compound, induced sweet potato detached leaf senescence and associated gene expression, and the effects were repressed by mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059. These data suggest that MAPK cascade is likely involved in ethylene signaling leading to leaf senescence and associated gene expression. With gene-specific primers and RT-PCR methods, a full-length cDNA, SPMAPK, was isolated from ethephon-treated sweet potato leaves. SPMAPK contained 1098 nucleotides (365 amino acids) in the open reading frame. Sweet potato SPMAPK also exhibited high amino acid sequence identities (ca. 79.8% to 83.4%) with plant MAPKs, and was most close to Arabidopsis MPK3 and MPK6 in phylogenetic tree analysis. RT-PCR analysis showed that SPMAPK gene expression was detected in roots, stems, and leaves. The mature and partial yellowing leaves expressed higher amount. SPMAPK gene expression was also inducible and significantly enhanced by ethephon. Results from studies with inhibitors or effectors showed that ethephon treatment resulted in acceleration of leaf senescence in detached sweet potato leaves, promotion of leaf chlorophyll content reduction and decrease of photochemical Fv/Fm, and induction of associated gene expression. These ethephon-mediated effects were all delayed or repressed by pretreatment with ethylene receptor inhibitor 1-methylcyclopropene (1-MCP), MAPK kinase inhibitor PD98059, NADPH oxidase inhibitor diphenyleneiodonium (DPI), antioxidant reduced glutathione, calcium ion chelator EGTA, and de novo protein synthesis inhibitor cycloheximide, respectively. Based on these results we conclude that an ethylene-inducible mitogen-activated protein kinase SPMAPK was isolated from sweet potato leaves, and expressed higher amount in mature and partial yellowing leaves. Ethephon-induced sweet potato SPMAPK expression was significantly repressed by 1-MCP, PD98059, DPI, reduced glutathione, EGTA and cycloheximide. These data also suggest that the possible signal components in ethephon-mediated leaf senescence and associated gene expression in sweet potato leaves likely include ethylene receptor, MAPK cascade, elevated H2O2 , external calcium influx, and de novo synthesized proteins. A possible ethylene signaling model leading to sweet potato leaf senescence and associated gene expression was also proposed.
論文審定書..............................................................................................i
誌謝............................................................................................................ii
圖次..........................................................................................................vii
縮寫表........................................................................................................x
中文摘要.................................................................................................xi
英文摘要...............................................................................................xiii
壹、緒論.......................................................................................................1
前言.......................................................................................................1
乙烯受器...............................................................................................3
MAPK cascade......................................................................................4
氧化壓力與抗氧化劑...........................................................................6
細胞外鈣離子流入...............................................................................7
貳、材料與方法.......................................................................................10
I. 實驗材料
A. 甘藷......................................................................................................10
B. 葉片老化相關基因..............................................................................10
C. 化學試劑..............................................................................................10
II. 實驗方法
A. PD98059對 ethephon 誘導甘藷葉片老化之影響............................10
藥品配製..............................................................................................11
PD98059前處理...................................................................................12
Ethephon 處理.....................................................................................12
葉綠素含量測量..................................................................................12
光合作用效率測量 (Fv/Fm value).....................................................13
DAB 染色分析....................................................................................13
H2O2定量分析.....................................................................................14
RT-PCR 分析.......................................................................................14
B. 從甘藷的老化葉片分子選殖 mitogen-activated protein kinase full length cDNA,並進行其胺基酸序列的 alignment 及親源樹分析..........................................................................................................18
甘藷 mitogen-activated protein kinase 全長 cDNA 分子選殖.......19
Sequence alignment 及親緣樹 (phylogenic tree) 分析.....................23
C. 甘藷葉片分離的 mitogen-activated protein kinase SPMAPK 之定性分析......................................................................................................24
SPMAPK 的時間 (temporal) 及空間 (spatial) 表現分析...............24
Ethephon 處理的 SPMAPK 表現之分析.........................................25
D. Inhibitors 與 effectors 對 ethephon 誘導甘藷葉片老化的影響..........................................................................................................25
PD98059、DPI、reduced glutathione、EGTA 及 cycloheximide 的前處理..................................................................................................25
1-MCP 的前處理................................................................................26
參、結果.....................................................................................................29
I. Ethephon 誘導甘藷葉片老化、H2O2含量上升及其老化相關基因的表現受 PD98059影響.........................................................................29
II. 甘藷葉片乙烯可誘導的 mitogen-activated protein kinase 分子選殖及定性分析..........................................................................................30
甘藷 mitogen-activated protein kinase 的分子選殖及生物資訊學分析..........................................................................................................31
甘藷 SPMAPK 的時間 (temporal) 及空間 (spatial) 表現分析.....33
Ethephon 增強甘藷 SPMAPK 基因的表現.....................................34
III. Ethephon 誘導甘藷葉片老化以及相關基因的表現受1-MCP、PD98059、DPI、reduced glutathione、EGTA 與 cycloheximide 影響..........................................................................................................36
1-MCP 減緩 ethephon 誘導甘藷葉片老化及降低其相關基因的表現..........................................................................................................36
PD98059減緩 ethephon 誘導甘藷葉片老化及降低其相關基因的表現..........................................................................................................37
DPI 減緩 ethephon 誘導甘藷葉片老化及降低其相關基因的表現..........................................................................................................39
Reduced glutathione 減緩 ethephon 誘導甘藷葉片的老化及降低其相關基因的表現..................................................................................40
EGTA 減緩 ethephon 誘導甘藷葉片老化及降低其相關基因的表現..........................................................................................................41
Cycloheximide 減緩ethephon 誘導甘藷葉片的老化及降低其相關基因的表現..........................................................................................43
肆、討論...................................................................................................45
參考文獻................................................................................................52

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