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研究生:尤曉莉
研究生(外文):shiou-li yu
論文名稱:小葉浮萍之色胺酸合成酵素beta鏈基因選殖與色胺酸及其衍生物受銅影響之化學分析
論文名稱(外文):Molecular analysis on copper-induced gene expression of TryptophanSynthase β- chain and the increased tryptophan and it’s derivatives mayimply anti-chemical stress mechanism in Lemna minor
指導教授:董啟功
指導教授(外文):CHII-GONG TONG
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生命科學研究所
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:中文
論文頁數:77
中文關鍵詞:色胺酸合成酵素浮萍色胺酸
外文關鍵詞:tryptophancopperduckweedHPLCRACETSB
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摘要
本論文之原點為小葉浮萍相減式cDNA 基因庫中受過量銅誘導
的基因-色胺酸合成酵素β鏈,色胺酸合成酵素β鏈是植物體合成色
胺酸過程中最後一個關鍵酵素,色胺酸在植物細胞中是唯一會轉化為
可吸收光能之物質的胺基酸,其下游的衍生物(tryptophan derivatives)
中較為熟知的有葉綠素(chlorophyll)、生長激素(auxin, IAA)、血清
素(serotonin)以及褪黑激素(melatonin)。其中,生長激素IAA 是
具有多功能的植物賀爾蒙,在細胞中作為訊息傳遞者或是基因的調節
者;褪黑激素則是首先發現於人類松果體之神經傳導物質,與光週期
有極高的相關性,並且具有清除自由基之能力。在植物界中,目前熟
知褪黑激素為種子中抵禦過氧化傷害之重要物質。
本論文利用5’端快速基因放大技術(5’-RACE)取得色胺酸合成
酵素β鏈之cDNA 全長,進一步取得基因上游之啟動子,以利日後分
析基因調節之機制,另外由南方氏點墨法判斷出色胺酸合成酵素β鏈
基因在浮萍基因組中應屬於一個中小型的基因家族(gene family)。
本論文同時使用高效液相層析儀追蹤浮萍在過量銅環境中,於不
同時間點下色胺酸與其重要衍生物之含量變化,證實色胺酸合成酵素
β鏈之基因表現與色胺酸及其重要衍生物之產生具有正相關性外,高
效液相層析儀同步自由基清除者偵測系統(HPLC on-line free radical
scavenger detection system)更有助於釐清此類衍生物在小葉浮萍中之
生理功能。
Abstract
Our previous study has identified several copper responsive genes in
duckweed, Lemna minor. One of the genes encodes tryptophan synthase
β-chain (TSB) which catalyzes the last step of tryptophan synthesis
pathway in plants. The strong expression of TSB under excess of copper
may indicate that the elevation of tryptophan synthesis and its subsequent
metabolic intermediates could respond to the toxicity imposed by this
metal. Tryptophan is a precursor of the phyto-hormone, auxin (IAA)
which mediates multiple signals in plants for development, gravitropism
and stress responses. Several tryptophan derivatives are also intrinsic
light-adsorbing compounds such as chlorophyll, serotonin and melatonin.
Melatonin was first discovered in pineal grain of the vertebrates that
governs the diurnal and photo-periodic response. Subsequently, it was
identified as a free radical scavenger in other organisms such as bacteria
and plants. This thesis correlated the timing of gene expression of TSB
and the accumulation profiles of the tryptophan and its derived
compounds such as IAA and melatonin in order to unfold the
physiological response to excess of copper. Genomic Southern analysis
showed TSB is a small- to mid-sized gene family. The full-length cDNA
was obtained by 5’-RACE and its upstream region was isolated using
genomic walking. Time course analysis for tryptophan and its derivatives
IAA and melatonin by HPLC showed reasonable correlation between
gene expression of TSB and the accumulation of the metabolites. The
further application by HPLC on-line free radical scavenger detection
system can assist the functional assays for such metabolites.
目錄
摘要----------------------------------------------------------------------------- Ⅰ
英文摘要----------------------------------------------------------------------- Ⅱ
目錄----------------------------------------------------------------------------- Ⅲ
圖表目錄----------------------------------------------------------------------- Ⅵ
附錄目錄----------------------------------------------------------------------- Ⅷ
縮寫與全名對照表----------------------------------------------------------- Ⅸ
壹、緒論----------------------------------------------------------------------- 1
一、前言-------------------------------------------------------------------- 1
二、銅在植物體內的生理特性----------------------------------------- 1
三、銅對植物體的毒害--------------------------------------------------- 2
四、植物抵禦過量銅毒害之策略-------------------------------------- 3
五、浮萍(Lemnaceae)簡介------------------------------------------- 3
六、色胺酸合成酵素β鏈基因與銅逆境下之浮萍------------------ 4
七、重要的色胺酸衍生物----------------------------------------------- 6
(1) 生長激素Indole-3-acetic acid ------------------------------ 7
(2) 褪黑激素Melatonin ------------------------------------------ 7
八、研究目的-------------------------------------------------------------- 8
IV
貳、材料與方法------------------------------------------------------------- 10
一、浮萍取得與馴養方式--------------------------------------------- 10
二、色胺酸合成酵素β鏈基因之分子生物基本研究------------- 10
1、金屬銅處理下總核糖核酸之萃取-------------------------- 10
2、重金屬處理之訊息核糖核酸純化與分析---------------- 11
3、小葉浮萍基因組去氧核糖核酸之萃取------------------- 12
4、南方氏點墨法------------------------------------------------- 13
5、cDNA 5’端快速複製法-------------------------------------- 16
6、基因上游啟動子之取得------------------------------------- 19
三、化學分析法追蹤色胺酸及其重要二次代謝物-------------- 21
1、小葉浮萍甲醇萃取物之製備------------------------------- 21
2、甲醇萃取物之固相萃取------------------------------------- 21
3、高效液相層析分析浮萍之甲醇萃取液------------------- 22
四、儀器設備------------------------------------------------------------ 25
五、試藥------------------------------------------------------------------ 25
參、結果--------------------------------------------------------------------- 27
一、南方氏點墨法分析------------------------------------------------ 27
二、cDNA 5’端快速複製法--------------------------------------------- 27
三、色胺酸合成酵素β鏈基因上游啟動子之取得---------------- 28
V
四、高效液相層析分析浮萍之甲醇萃取物------------------------ 29
五、清除自由基能力之測定------------------------------------------ 29
肆、討論--------------------------------------------------------------------- 32
伍、建議--------------------------------------------------------------------- 37
陸、參考文獻--------------------------------------------------------------- 38
圖、表------------------------------------------------------------------------ 44
附錄-------------------------------------------------------------------------- 59
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