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博碩士論文 etd-0621116-163010 詳細資訊
Title page for etd-0621116-163010
論文名稱
Title
甘藷絲裂原活化蛋白激酶SPMAPK參與在ethephon誘導的葉片老化過程
Sweet potato mitogen-activated protein kinase SPMAPK participates in ethephon-mediated leaf senescence
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
66
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2016-07-19
繳交日期
Date of Submission
2016-07-21
關鍵字
Keywords
甘藷、益收生長素、PD98059、絲裂原活化蛋白激酶、葉片老化
Sweet potato, Ethephon, PD98059, Mitogen-activated protein kinase, Leaf senescence
統計
Statistics
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The thesis/dissertation has been browsed 5681 times, has been downloaded 32 times.
中文摘要
Ethephon為一種可以釋放植物生長調節物質乙烯的化合物,被廣泛應用於農業上,並且與促進葉片老化有關,其中絲裂原活化蛋白激酶 (mitogen-activated protein kinase; MAPK) 已被證實參與阿拉伯芥的乙烯訊息傳導途徑,然而其在甘藷葉片老化過程所扮演的角色並不清楚。本實驗室先前從ethephon處理之甘藷葉片選殖出一段全長的絲裂原活化蛋白激酶SPMAPK cDNA,其open reading frame含有1,098個核甘酸(365個胺基酸),與植物絲裂原活化蛋白激酶胺基酸序列比對具有高相似性 (79.8% 至83.4%),親源樹分析結果顯示,與阿拉伯芥的MPK3/MPK6序列相似度最高。將對應於SPMAPK成熟蛋白質的cDNA部分構築於pET30a表現載體,以誘導、純化其融合蛋白質並製備多株抗體及作為實驗的處理來源。Ethephon處理72小時顯著促進甘藷葉片老化、增加SPMAPK的表現量及老化相關指標表現 (包括葉片黃化、葉綠素含量和Fv/Fm下降及NO/H2O2/MDA含量上升),前處理MAPK cascade抑制劑PD98059可呈劑量依賴性,有效延緩ethephon誘導的葉片老化、SPMAPK表現量及老化相關指標表現。MAPK cascade抑制劑PD98059對ethephon的影響可經由外加純化的SPMAPK融合蛋白質而受抑制抵銷並回復至ethephon單獨處理的葉片老化狀態。而外加SPMAPK融合蛋白之效果亦可經由外加前,前處理此SPMAPK融合蛋白質於 (a) 950C下加熱五分鐘或 (b) 室溫下與anti-SPMAPK抗體混合靜置兩小時而受抑制抵銷並回復至PD98059前處理的葉片老化程度。單獨外加SPMAPK融合蛋白質於ethephon處理 (不是黑暗處理)的甘藷葉片亦可顯著促進葉片老化。根據上述結果推論甘藷SPMAPK為ethephon可誘導的絲裂原活化蛋白激酶,並且參與在ethephon訊息傳導途徑,促進甘藷葉片老化。
Abstract
Ethephon, an ethylene-releasing compound, is widely used in agriculture and promotes leaf senescence. Mitogen-activated protein kinase (MAPK) has been reported to act as a signal component of ethylene in Arabidopsis thaliana, however, is unclear in sweet potato. Recently, a full-length cDNA, SPMAPK, has been cloned from sweet potato leaves and encodes a putative MAPK, which contains 1,098 nocleotides (365 amino acids) in its open reading frame and exhibits high amino acid sequence identity (79.8–83.4%) with plant MAPKs. Phylogenetic tree analysis shows that it displays the most closely-associated relationship with Arabidopsis MPK3/MPK6. The cDNA corresponding to the SPMAPK mature protein was constructed with pET30a expression vector for recombinant fusion protein purification, polyclonal antibody production, and exogenous application experiments. Ethephon treatment for 72 h significantly promoted leaf senescence, elevated SPMAPK expression, and altered senescence-associated marker levels (including morphological leaf yellowing, reduction of chlorophyll and Fv/Fm contents, and elevation of NO/H2O2/MDA levels), which could be attenuated dose-dependently by the pre-treatment of MAPK cascade inhibitor PD98059. Effects of PD98059 can be repressed by the exogenous SPMAPK fusion protein and reversed to a senescent level similar to the ethephon alone. Pre-treatment of SPMAPK fusion protein prior to its application with (a) 950C for 5 min or (b) the anti-SPMAPK antibody at 250C for 2 h abolished its repressive effects and reversed to a senescent level similar to the PD98059 treatment. In addition, exogenous SPMAPK fusion protein did promote ethephon, but not dark, mediated leaf senescence. These data conclude that SPMAPK is an ethephon-inducible mitogen-activated protein kinase and participates in its signal transduction pathway leading to leaf senescence in sweet potato.
目次 Table of Contents
目 錄
論文審定書…………………………………………………………..… i
誌謝………………………………………………………………….… ii
中文摘要………………………………………………………….…... iii
英文摘要………………………………………..…………………….. iv
目錄…………………………………………………………….……… v
圖次………………………………………………………………..….. viii
縮寫表……………………………………………………………….… x
壹、緒論
甘藷………………………………………………………………… 1
葉片老化…………………………………………………………… 1
乙烯的生合成及訊息傳導途徑…………………………………… 3
乙烯受器途徑……………………………………………………… 4
H2O2/NADPH oxidase及Ca2+/calmodulin 訊息傳導途徑….…… 5
NO訊息傳導途徑…………………………………………………. 6
MAPK cascade訊息傳導途徑…………………………………….. 6
研究動機與目的…………………………………………………… 7
貳、材料與方法
一、材料方法………………………………………………….… 9
甘藷…………………………………………………………. 9
甘藷相關基因及抗體………………………………………. 9
化學試劑……………………………………………………. 9
二、實驗方法…………………………………………………… 10
Ethephon處理………………………………………………. 11
PD98059前處理……………………………………………. 11
SPMAPK 融合蛋白處理……………………...…………… 12
PD98059及SPMAPK融合蛋白處理……………………... 13
重組 SPMAPK融合蛋白構築、表現及純化…………….. 13
SDS-PAGE電泳分析………………………………………. 16
葉片外觀形態分析…………………………………………. 19
葉綠素含量測量……………………………………………. 19
Fv/Fm測量…………………………………………………. 19
DAB染色…………………………………………………... 20
H2O2含量測量……………………………….……………... 20
NO含量測量……………………………………………….. 21
MDA含量測量…………………………………………….. 21
RT-PCR分析……………………………………………….. 22
參、結果
Ethephon誘導葉片老化與SPMAPK表現量有正相關………... 26
MAPK cascade抑制劑PD98059延緩ethephon誘導甘藷葉片老化及降低SPMAPK基因表現量……………………………………... 26
重組 SPMAPK 融合蛋白質的誘導表現與純化……………… 27
外加 SPMAPK融合蛋白抑制PD98059影響並且回復ethephon促進葉片老化作用………………………………………………… 28
外加 SPMAPK融合蛋白對黑暗誘導葉片老化無顯著影響…. 29
肆、討論……………………………………………………………... 30
伍、參考文獻………………………………………………………... 34
參考文獻 References
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