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研究生:洪景祥
論文名稱:影響杜鵑類菌根菌及暗色隔膜內生真菌分泌漆氧化酵素的因子
論文名稱(外文):Affecting Factors of Laccase Secretion from Ericoid Mycorrhizal Fungi and Dark Septate Endophytic Fungi
指導教授:林瑞進林瑞進引用關係
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:森林暨自然資源學系研究所
學門:農業科學學門
學類:林業學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:81
中文關鍵詞:暗色隔膜內生真菌分解能力杜鵑類菌根菌漆氧化酵素
外文關鍵詞:dark septate endophytic fungidecomposition abilityericoid mycorrhizal fungilaccase
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杜鵑花屬(Rhododendron)植物一般棲息在石南原的特殊地形,該地形的枯落物具備葉厚且富含革質的特性,植物難以透過酵素完全分解其枯落物,也因此造成枯落物長年累積形成石南原的特殊地形,然而杜鵑花屬植物能與杜鵑類菌根菌與暗色隔膜內生真菌共生,並透過該兩類菌株分解酵素,幫助植體在石南原地中生存。本試驗欲探討天數及濃度因子,何者較能促進杜鵑類菌根菌及暗色隔膜內生真菌分泌漆氧化酵素,並比較各菌種間差異。本試驗將本研究室分離出的有益內生菌,以及向國外菌種中心購買的菌株,共10支菌株(6支屬杜鵑類菌根菌,4支屬暗色隔膜內生真菌;皆已證實能和杜鵑屬植物共生)進行能分解木質素的漆氧化酵素定性試驗,篩選出具有其酵素能力的菌株;再將挑選出的4 支菌株(Rher、Rhy3、CkDB5及Rk1)挑出,並控制水萃液濃度及天數來探討菌株在不同天數及水萃液濃度下漆氧化酵素活性。
首先,於酵素定性試驗中根據能分泌漆氧化酵素之菌株,分別為Rher、Rhy3、CkDB5及Rk1 ,此4支菌株具有漆氧化酵素活性菌株。在漆氧化酵素定量試驗中,不同菌株在不同濃度的水萃液中漆氧化酵素表現也有所不同,其中Rher菌株在本試驗中漆氧化酵素活性表現最佳,其值高達 5.13 U/mL,其次為Rhy3菌株,值為 5.01 U/mL,且觀察到菌株的酵素分泌量於10%及50%的水萃液濃度中會隨著天數增加而提升,於10%水萃液濃度下普遍於第21天產生最大值後下降,於50%水萃液濃度下於第21天產生最大值後下降。綜合上述試驗結果證實,Rhy3、Rher、CkDB5及Rk1 4支菌株具有分泌漆氧化酵素之能力;此4支菌株於10%濃度中會隨著天數遞增而提升漆氧化酵素分泌量,且不同菌株於同天數及同濃度下所分泌的漆氧化酵素產量也有所不同,其中以Rher分泌量為4支菌中最佳。
綜合上述發現菌株、天數及濃度三者因子皆對漆氧化酵素分泌量呈現顯著差異,證實三因子皆會影響漆氧化酵素分泌量。透過漆氧化酵素定量試驗證實,菌株所分泌的漆氧化酵素最大值普遍出現在第28天100%水萃液濃度中,且普遍趨勢為隨著天數增加,酵素活性達到最大值後開始下降,然而也有出現先下降後上升的趨勢,研判為分解大分子時產生的現象,而其生物量跟水萃液的酚類濃度變化是否與酵素活性量呈現相關性都值得後續探討。
Rhododendron plants generally inhabit the special terrain of the heather. The litter of this terrain has the characteristics of leafy and leathery, and the plants cannot completely decompose their litter through their own enzymes, thus causing the accumulation of litter for many years. The special topography of heathland, it is difficult for Rhododendron plants to coexist with ericoid mycorrhizal fungi and dark septate endophytic fungi, and through these two strains to decompose enzymes, help the plants survive in poor nutrient, and this experiment and to explore the factors affecting the production of enzymes by ericoid mycorrhizal fungi and dark septate endophytic fungi; therefore, this experiment firstly explored the beneficial endophytic fungus isolated from the laboratory, a total of 10 strains (6 strains of the ericoid mycorrhizal fungi, 4 strains of dark septate endophytic fungi; proven to be symbiotic with Rhododendrons plants; qualitative test for decomposition of laccase enzymes; and selection of symbiotic strains with the ability to secrete multiple enzymes; to explore the ability of these strains to laccase activity, for different concentrations and times.
The extracted 10 strains first underwent the affinity test via the secretory enzyme assays, 4 strains (Rhy3, Rher, CkDB5, Rk1 strains) capable of secreting laccase and lignases were selected. In the laccase activity test, the laccase activity of different strains in different concentrations of litter extracts were also different. The Rher strain has the best performance at as high as 5.13 U/mL, followed by Rhy3 strain with a value of 5.01 U/mL. It was observed that the enzyme secretion of the strain increased in accordance to the number of days and gradually decreased after the maximum value was produced.
Based on the above findings, the strains, days, and concentrations showed very significant differences in the amount of laccase activity (p <0.01), which confirmed that all three factors will affect the amount of laccase activity secretion; and through median analysis and graphs, it was concluded that at high concentrations the laccase activity of strains is generally better, but whether the changes in biomass and phenol concentration are related to enzymes is worth further discussion.
摘要 I
Abstract III
目錄 V
圖目錄 VIII
Ⅰ、前言 1
Ⅱ、前人研究 2
(I) 菌根菌概述 2
1. 杜鵑類菌根菌 4
2. 暗色隔膜內生真菌(DSEF) 4
(II) 杜鵑類菌根之效益 6
1. 抗重金屬能力 6
2. 幫助宿主植物在養分貧瘠地生長 6
(1) 氮對植物的影響 6
(2) 杜鵑類菌根菌氮源的利用情形 7
3. 分解能力 8
(III) 暗色隔膜內生真菌之效益 9
1. 分解能力 9
2. 幫助宿主植物生長 10
3. 增加宿主植物抗逆境 10
(IV) 有機質分解及養分利用 11
1. 漆氧化酵素 12
2. 漆氧化酵素定性及定量 13
(V) 真菌對於分泌漆氧化酵素之因子 15
1. 不同介質 15
2. 不同菌種及天數 15
3. 不同誘導物濃度 16
III、研究目的 16
Ⅳ、研究材料與方法 17
(I) 試驗流程圖 17
(II) 試驗材料 18
1. 菌株 18
2. 枯落物 19
(III) 菌株分泌酵素測定 19
1. 漆氧化酵素 19
2. 過氧化酵素 19
3. 纖維素酵素 19
(IV) 菌株在不同水萃液濃度及天數之漆氧化酵素活性 20
1. 菌株培育 20
2. 不同水萃液濃度及天數之漆氧化酵素活性 20
3. 漆氧化酵素活性量測 20
(V) 分析方法 21
1. 變異數分析 21
V、結果 22
(I) 菌株分泌酵素測定 22
(II) 各因子間交互作用 24
1. 各菌株在不同濃度水萃液及天數下之漆氧化酵素活性比較 25
(1) Rher菌株漆氧化酵素定量 26
(2) Rhy3菌株漆氧化酵素定量 30
(3) CkDB5菌株漆氧化酵素定量 34
(4) Rk1菌株漆氧化酵素定量 38
2. 菌株在不同天數下漆氧化酵素活性表現 42
(1) 菌株培育第14天活性表現 42
(2) 菌株培育第21天活性表現 44
(3) 菌株培育第28天活性表現 45
3. 菌株在不同濃度下漆氧化酵素活性表現 47
(1) 菌株於10%水萃液濃度下表現 47
(2) 菌株於50%水萃液濃度下表現 48
(3) 菌株於100%水萃液濃度下表現 50
VI、討論 52
(I) 菌株分泌酵素測定 52
(II) 菌株在不同水萃液濃度及天數之漆氧化酵素活性 52
VII、結論 59
VIII、引用文獻 61
IX、附錄 75
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