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研究生:魏麟
研究生(外文):Lin Wei
論文名稱:細菌吲哚乙醯亮胺酸水解酵素定性、基因偵測與探討吲哚乙酸對細菌之影響
論文名稱(外文):Characharacterization, gene detection of bacterial IAA-Leucine hydrolase and study of the effect by IAA on the bacteria
指導教授:周志青
指導教授(外文):Jyh-Ching Chou
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
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:2009
畢業學年度:98
語文別:中文
論文頁數:75
中文關鍵詞:細菌水解酵素
外文關鍵詞:IAA conjugate hydrolaseIAAbacteria
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Indole-3-acetic acid (IAA)為自然界高等植物中,最主要的植物生長激素,主要由頂端分生組織、幼葉、發育中的果實或種子中生成。在植物中的生理作用如下:增加植物細胞壁延展性以促使細胞的體積的伸展、頂端優勢、誘導生成側根及不定根、可與細胞分裂素(cytokinin)用於植物組織培養、促進維管束組織分化等。而在植物體內,IAA常以共軛物的形式存在以調節IAA在植物體內的活性與作用,例如:IAA的儲存與再利用與降解、保護IAA避免受到酵素攻擊與IAA在維管束內長程運輸。目前對於IAA調控植物的機制尚未十分清楚,而IAA醯胺類共軛物在IAA生理代謝中,扮演關鍵性的中間產物,研究IAA醯胺類共軛物水解酵素,有助於了解IAA在植物生理中的調控機制。然而在植物體中大部分IAA共軛物水解酵素專一性都不高,所以本研究以細菌為研究對象,希望從自然生態環境中,找到可以產生IAA-leucine hydrolase的細菌株,並觀察IAA對於細菌株生長的影響。首先利用集菌法(enrichment),從海灘上雜草根部附著土壤樣品中,以N-acetyl-D,L-leucine做為唯一的碳源與氮源,培養出數個細菌株,再以TLC酵素活性分析篩選出編號7-8-1-1細菌,7-8-1-1為革蘭氏陽性菌,打破細胞所收集的初萃取液具有專一性水解IAA-Leu (N-[3-indolylacetyl]-L-leucine)的能力,最理想反應pH值為6.5~7.5之間,但不需要誘導物在正常生長狀態下便可以產生此酵素活性,IAA或是其他具auxin生理活性的分子則會抑制7-8-1-1的生長繁殖。藉由這些研究,提供了一個新的菌種與indole-3-acetyl-L-leucine hydrolase做為實驗的工具,有利於將來研究IAA-Leu對植物體生長發育的影響。
Indole-3-acetic acid (IAA) is the major auxin in higher plants. It is synthesized in shoot apical meristems, young leaves, and developing fruits and seeds. The developmental effects of IAA include increasing the extensibility of the cell wall for cell elongation, apical dominance, promotion of lateral and adventitious roots, interaction with cytokinin for plant tissue culture and induction of vascular differentiation. Most of IAA exists as conjugated forms in plants for multiple functions, such as storing, subsequent reuse and homeostatic control of IAA, protecting free IAA from enzymatic destruction and long distance transport of IAA in vascular tissue. The metabolic mechanism of IAA in plants is far from clear, but IAA amino acid conjugates play a key intermediate in IAA-controled plant physiology and metabolism. The study of IAA amidohydrolases can help us to understand the mechanism of IAA in plant. We intended to isolate bacterial strains exhibiting significant IAA-leucine hydrolases activity from bacteria since IAA-amino acid conjugate hydrolase identified from plants were not substrate specific. We also measured the effects by IAA on the growth of the bacterial strain. First, by standard enrichment, we used N-acetyl-D, L-leucine as the sole source of carbon and nitrogen sources to cultivate a number of bacterial strains that is from the rhizosphere of weeds on the beach. The bacterial strain, labeled 7-8-1-1, was selected by TLC analysis. The strain 7-8-1-1 was a gram-positive bacterium. The crude extract of 7-8-1-1 had substrate specificity to hydrolyze IAA-Leu (N-[3-indolylacetyl]-L-leucine). The optimal pH was 6.5~7.5. It was able to produce this enzyme activity in normal growth condition without inducer, but IAA and other compound, which had the physiological activity, could inhibit the growth and reproduction of 7-8-1-1. In summary, we identified a new bacterial strain with indole-3-acetyl-L-leucine hydrolase activity which may provide a tool for experiments that will help future studies of IAA-Leu on the growth and development of plants.
中文摘要---------------------------------------------------------------------------I
Abstract---------------------------------------------------------------------------III
縮寫表-----------------------------------------------------------------------------V
目錄------------------------------------------------------------------------------VII
圖表目錄--------------------------------------------------------------------------X
一、前言-----------------------------------------------------------------------------1
1. IAA--------------------------------------------------------------------------1
2. IAA在植物體中的生理作用-------------------------------------------1
3. IAA的合成與降解--------------------------------------------------------3
4. IAA共軛物-----------------------------------------------------------------4
5. IAA共軛物水解酵素----------------------------------------------------6
二、目的---------------------------------------------------------------------------9
三、材料方法--------------------------------------------------------------------11
1. 細菌株收集--------------------------------------------------------------11
2. 未知細菌株初萃取液製備--------------------------------------------11
3. 未知酵素TLC活性測試---------------------------------------------12
4. 革蘭氏菌種鑑定-------------------------------------------------------13
5. IALEUH在BSM液態培養誘導測試------------------------------13
6. 酵素最佳反應pH值測試--------------------------------------------14
7. 酵素專一性測試-------------------------------------------------------14
8. 7-8-1-1在不同誘導物下的生長速率測定-------------------------14
9. 7-8-1-1在不同濃度IAA下的生長速率---------------------------15
10. 7-8-1-1在不同種類auxin下的生長速率------------------------15
11. Degenerate PCR-------------------------------------------------------15
12. ialeuH基因探針設計------------------------------------------------16
13. 南方墨點法偵測ialeuH基因片段--------------------------------17
四、結果-------------------------------------------------------------------------19
1. 菌種收集與革蘭氏染色結果----------------------------------------19
2. 酵素誘導測試----------------------------------------------------------19
3. 酵素最佳反應pH值測試--------------------------------------------19
4. 酵素專一性測試-------------------------------------------------------20
5. 7-8-1-1在不同誘導物下的生長速率測定-------------------------20
6. 7-8-1-1在不同濃度IAA下的生長速率測定---------------------20
7. 7-8-1-1在不同種類auxin下的生長速率--------------------------21
8. Degenerate PCR---------------------------------------------------------21
9. ialeuH基因探針設計--------------------------------------------------21
10. 南方墨點法偵測ialeuH基因片段--------------------------------21
五、討論-------------------------------------------------------------------------23
六、參考文獻-------------------------------------------------------------------29
七、圖表資料-------------------------------------------------------------------35
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