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研究生:袁秋英
研究生(外文):Chiou-Ing Yuan
論文名稱:華九頭獅子草對嘉磷塞之生理生化反應及耐藥性機制探討
論文名稱(外文):Physiological, biochemical responses and tolerant mechanism of Dicliptera chinensis against glyphosate
指導教授:陳益明陳益明引用關係
指導教授(外文):Yih-Ming Chen
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:植物學研究所
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:157
中文關鍵詞:嘉磷塞除草劑耐藥性華九頭獅子草EPSPS酵素
外文關鍵詞:glyphosateherbicide toleranceDicliptera chinensisEPSP synthase
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華九頭獅子草(Dicliptera chinensis Juss.)為爵床科草本植物,分佈於台灣全省低海拔地區,此植物對於除草劑嘉磷塞(glyphosate)具高度耐性,經由此藥劑測定,華九頭獅子草之ED50值為紫花霍香薊、野甘草、野莧、鬼針等種雜草之2-7倍。耐藥特性為華九頭獅子草之原有特質,可遺傳於子代。噴施20 mM嘉磷塞造成華九頭獅子草葉片生長速率減慢,幼葉葉色淡化,葉肉柵狀組織及海綿組織,呈現不規則緊密排列,導管及篩管數目減少。但是施藥後7日,幼葉細胞之細胞核、葉綠體、粒線體及澱粉粒等皆完整。以14C-嘉磷塞測試,施藥後6小時藥劑即進入植體,施藥後168小時,根部累積量最大,嘉磷塞於華九頭獅子草及紫花霍香薊-嘉磷塞敏感植物之吸收與傳導率相似。施藥後14日,植體內無嘉磷塞代謝物-aminomethylphosphonic acid (APMA)之檢出,顯示華九頭獅子草之耐藥特性與藥劑代謝無顯著關聯性。20 mM 嘉磷塞處理後14日,華九頭獅子草幼葉總胺基酸含量因leucine及phenylalanine增加而增高,成熟葉之phenylalanine、serine、proline及methionine及根部phenylalanine含量略增加。華九頭獅子草之幼葉、成熟葉及根部shikimic acid含量於施藥後增加為未施藥者之5-9倍。幼葉及成熟葉二次代謝物benzoic acid、chlorogenic acid 及t-cinnamic acid含量增加1.5至3倍。幼葉及根部之protocatechuic acid 含量於施藥後14日增加為未施藥者之2.5及6.5倍。華九頭獅子草之5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)活性以幼葉最高。華九頭獅子草及紫花霍香薊幼葉噴施嘉磷塞,二者EPSPS之I50值分別為6.8 mM及0.8 mM。藥劑處理後24.5小時,華九頭獅子草幼葉仍具有活性,紫花霍香薊者已喪失活性。華九頭獅子草EPSPS具二個cDNAs:EPSPS-1及EPSPS-2 (1891 bp, open reading frame為1548 bp,transit peptide為210 bp),二者差異為26個鹼基不同。華九頭獅子草EPSPS胺基酸與矮牽牛、菸草、番茄及阿拉伯芥者具84-88%相似度。此植物EPSPS mRNA之表現以幼葉最強,其次為花、根及莖部。嘉磷塞處理後8-24小時,華九頭獅子草EPSPS mRNA表現明顯增加,以16小時表現量最多。紫花霍香薊EPSPS mRNA於 8小時後即大量降低。華九頭獅子草 mature EPSPS蛋白質含量於2 mM嘉磷塞浸藥處理後8.5小時開始增加,至16.5小時增加為未施藥者之55倍。這些證據顯示華九頭獅子草對嘉磷塞之耐藥特性有二原因:一、華九頭獅子草具高活性之EPSPS。二、經由嘉磷塞處理後,可誘導華九頭獅子草EPSPS mRNA之表現並使EPSPS蛋白質含量增加。

Dicliptera chinensis Juss. is a naturally occurring glyphosate-tolerant plant. It is an indigenous Acanthaceae of Taiwan and East Asia. In the last two decades, this plant has become a dominant weed in some of the lowland orchards in central Taiwan as a result of chemical weed control. D. chinensis survived after spraying with 3.3 kg ha-1 of glyphosate. Greenhouse tests showed that ED50 for D. chinensis was 2-7 times higher than that for other common weeds species. This tolerance of D. chinensis to glyphosate was closely related with it’s developmental stages. The tolerance of D. chinensis to glyphosate was not derived from selection by repeated use of this herbicide. There was no glyphosate-sensitive D. chinensis variety in Taiwan. The absorption, translocation and degradation of glyphosate in D. chinensis were similar to Ageratum houstonianum, suggested that these were not the basis of the tolerance. The accumulation of shikimate in young leaf was increased 5- to 9-fold in leaf and root tissues. The levels of phenylalanine, tyrosine and tryptophan in young leaves, and phenylalanine in mature leaves were not decreased at 14 day after treatment. These changes were different from those of glyphosate-sensitive plants. Protocatechuate (PCA) levels were 2.5- and 6.5-fold higher than those of the control in young leaves and roots. The less accumulation of shikimate, PCA, gallic acid and increase of aromatic amino acids in herbicide-treated D. chinensis plants could explain that the shikimate pathway of this plant did not completely be blocked by glyphosate. In comparison to the glyphosate-sensitive weed A. houstonianum, D. chinensis had higher 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity even before glyphosate treatment. Glyphosate treatment resulted in significant increase of the EPSPS mRNA and protein level. This increase was apparent at about 8 h after glyphosate treatment, and peaked at about 16 h. The higher endogenous EPSPS activity and the induction by glyphosate could probably explain the tolerance to glyphosate. Two cDNAs representing two distinct EPSPS genes were isolated. Their sequences were highly homologous to the known plant and bacterial EPSPS. Northern and western blot analyses revealed differential tissue distribution. The analysis of mRNA and protein expression also suggested possible posttranscriptional regulation.

目 錄
中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………3
縮寫列表…………………………………………………………………5
第一章 前言
一、 概說:華九頭獅子草與嘉磷塞………………………………..7
二、 嘉磷塞之作用機制……………………………………………..8
三、 嘉磷塞於植體之吸收、傳導與代謝…………………………10
四、 嘉磷塞對植物形態、胺基酸、shikimic acid…………….…..15
及二次代謝物之影響
五、 嘉磷塞之抗藥性及EPSPS 之特性…………………………..18
第二章 材料與方法
一、 華九頭獅子草生長與嘉磷塞之劑量反應關係………………..23
二、 嘉磷塞於華九頭獅子草之吸收傳導與代謝…………………..25
三、 嘉磷塞對華九頭獅子草葉片組織及胞器之影響……………. 30
四、 嘉磷塞對華九頭獅子草生理生化反應之影響………………. 36
五、 華九頭獅子草之EPSPS 基因與其蛋白質特性……………….44
第三章 結果
一、華九頭獅子草與嘉磷塞之劑量反應關係……………………….61
二、嘉磷塞於華九頭獅子草之吸收傳導與代謝…………………….63
三、嘉磷塞對華九頭獅子草形態、生理生化反應之影響………….64
四、華九頭獅子草之EPSPS 基因與其蛋白質特性…………………69
第四章 討論…………………………………………………………….73
圖列……………………………………………………………………...88
表列…………………………………………………………………….113
參考文獻……………………………………………………………….125
附錄…………………………………………………………………….146

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