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研究生:呂仲祥
研究生(外文):Chung-Hsiang Lu
論文名稱:利用基因剔除技術初步探討胞外幾丁質外切酵素基因 exc1 及 exc2 在木黴菌 (Trichoderma harzianum) 對 Rhizoctonia solani 拮抗能力之影響
論文名稱(外文):Exploiting gene knock-out technique to preliminarily study the influences of extracellular exochitinase genes exc1 and exc2 in antagonistic activity of Trichoderma harzianum against Rhizoctonia solani
指導教授:林世斌林世斌引用關係
指導教授(外文):Lin, Shih-Bin
口試委員:林國知郭村勇陳瑞祥林世斌
口試委員(外文):Lin, Kuo-ChihKuo, Tsun-TungChen, Ruey-ShyangLin, Shih-Bin
口試日期:2011-08-19
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物技術研究所碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:97
中文關鍵詞:木黴菌超寄生幾丁質酵素同源重組基因剔除農桿菌
外文關鍵詞:TrichodermaAgrobacterium tumefaciensmycoparasitismchitinaseshomologous recombinationgene knock out
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木黴菌屬 (Trichoderma spp.) 廣泛存在於自然界,因其對多種植物病原真菌具有拮抗能力 (antagonism),故成為多種植物病害的生物防治製劑 (biocontrol agent)。真菌超寄生機制 (mycoparasitism) 是木黴菌對病原真菌拮抗作用中之主要機制之一,包括趨化性 (chemotactic) 生長、接觸、識別、纏繞及穿透 (penetration) 等過程。該機制中,咸認為木黴菌分泌之兩種幾丁質外切酵素 (exochitinase, EXC1 and EXC2) 可能扮演重要角色,但機制尚不清楚。因此本研究以基因剔除技術探討 exc1 與 exc2 在 Trichoderma harzianum 與植物病原真菌 Rhizoctonia solani 拮抗過程中扮演之角色。本實驗先以 iPCR (Inverted PCR) 技術,選殖 T. harzianum (BCRC 30821) 之 exochitinases 基因 (exc1 與 exc2) 編碼區外側之基因序列(兩端各約1.5 kb),構築阻斷載體 (disruption vectors, pKo-exc1 & pKo-exc2),利用農桿菌媒介轉形法 (Agrobacterium tumefaciens-mediated transformation, ATMT) 藉同源重組基因置換原理,各別剔除 T. harzianum 之 exc1 及 exc2 基因。透過菌株培養以及與植物病原真菌對峙培養實驗發現, exc1 基因的剔除會改變 T. harzianum 之生長形態、降低生長速率以及失去對 R. solani (BCRC 31252) 之拮抗行為;利用光學顯微鏡觀察發現,其可能喪失超寄生能力。而 exc2基因的剔除可能促進 T. harzianum 對 R. solani 之拮抗行為與超寄生能力,但不影響其生長形態與速率。
Fungal species of the genus Trichoderma occur worldwide and can antagonize against several economically important plant pathogenic fungi and are therefore used as biocontrol agents against several plant pathogenic fungi. Mycoparasitism, one of the main antagonizing mechanisms that Trichoderma utilizes in direct confrontation with fungal pathogens, involves complex procedure including chemotropism, contact, lectin-mediated recognition, and formation of trapping and penetration structures. Two exochitinases (EXC1 and EXC2), which are partially produced before direct contact with the host, may play major roles in the process, but mechanism is still unclear. In this study, we exploited gene knock-out technique to study the influence of extracellular exochitinase genes (exc1 and exc2) in antagonistic activity of T. harzianum against fungal pathogens Rhizoctonia solani. The DNA fragments flanked (~ 1.5 kb each side) of the T. harzianum (BCRC 30821) exochitinase genes (exc1 and exc2) were obtained by inverse PCR and subsequently used for constructing disruption vector (pKo-exc1 & pKo-exc2). These two vectors, using as gene-replacement cassettes, were introduce by Agrobacterium tumefaciens-mediated transformation (ATMT) into T. harzianum to obtain gene knock out strains, Δexc1 and Δexc2 strains. The confrontation test against R. solani demonstrated the growth rate, colony morphology, and antagostic activity of Δexc1 strain were significantly affected. Microscopic inspection of Δexc1 reveled that Δexc1 strain lost its mycoparasitic activity.On contrast Δexc2 strain displayed no difference in the growth rate and colony pattern but slightly enhanced in its antagonistic activity
目 錄
摘要…………………………………………………………………………... I
英文摘要…………………………………………………………………….. II
目錄…………………………………………………………………………. III
圖目錄……………………………………………………………………… VII
附錄目錄……………………………………………………………………. IX
壹、前言……………………………………………………………………... 1
貳、文獻整理………………………………………………………………... 3
一、Chitinases…………………………………………………………….. 3
(一)Chitinases 的分類……………………………………………… 3
二、Chitinases 與真菌細胞壁之重塑 (remodeling)…………………..... 5
三、木黴菌………………………………………………………………. 10
四、木黴菌的生防機制 (Biocontrol)……………………………...…… 11
(一)超寄生作用 (Mycoparasitism)……………..………………… 11
(二)競爭作用 (Competition)……………………………………… 14
(三)抗生作用 (Antibiosis)…………….………………………….. 15
五、T. harzianum 之 chitinases 與生物防治的關聯………………….. 16
六、農桿菌媒介的絲狀真菌遺傳轉形 ( Agrobacterium tumefaciens-
mediated transformation, ATMT )………………………………….. 19
(一)ATMT 法的分子機制………………...……………………… 20
(二)ATMT 法之轉形特點………………………...……………… 22
(三)影響 ATMT 媒介真菌轉形效率的因素………….………… 23
(四)標靶突變 (Targeted mutagenesis) 與 ATMT………………. 26
七、研究目的……………………………………………………………. 29
参、實驗架構流程圖………………….…………………………………… 31
肆、材料與方法…………….……………………………………………… 32
一、 儀器設備…………………………………………………………... 32
二、 exc1 與 exc2 基因 ORF 編碼區完整序列之獲得……….……. 32
(一)T. harzianum 之基因組 DNA 的萃取……………………… 33
(二)DNA 聚合酵素連鎖反應與瓊膠電泳分析…………..……… 34
1. DNA 聚合酵素連鎖反應………..…………………………….. 34
2. DNA 電泳分析……………………………………………….... 34
(三)瓊膠電泳萃取 DNA 與 TA cloning 反應……….………… 35
1. 瓊膠電泳萃取 DNA…………………………………………... 35
2. TA cloning 反應…………………….…………………………. 36
(四)E. coli DH5α 勝任細胞的製備與質體 DNA 之轉形作用.… 36
1. 氯化鈣法製備勝任細胞……………………………………..… 36
2. 大腸桿菌的轉形作用……………………………………..…… 37
(五)小量質體 DNA 之製備……………………………………… 37
三、以 Inverted PCR 技術選殖已知基因序列兩側外之基因序列...… 38
四、構築 Disruption vector……………………………………………... 39
五、同源重組 (Homologous recombination)…………………...…...…. 43
(一)農桿菌之電破法勝任細胞製備……………….……...……… 43
(二)農桿菌的電破法 (Electroporation) 轉型作用….…………… 43
(三)ATMT 法轉形 T. harzianum………………………………… 43
六、Gene knockout strains 之確認………………………………...…… 44
七、對峙培養實驗 (Confrontation Assays)……………………………. 45
伍、結果與討論……………………………………………………………. 46
一、Gene knockout 所需之 homologous sequence 的選殖與製備…… 46
(一)exc1 與 exc2 編碼區完整基因序列之獲得與分析………… 46
(二)利用 iPCR 獲得編碼區基因轉譯起始碼上游之基因序列… 47
二、ATMT 法轉形 T. harzianum 與同源重組……………………..… 47
三、Gene knockout strain 之確認……………………………………… 48
四、Gene knockout strains 之生長特性………………………………... 49
五、Gene knockout strain 與 R. solani 之對峙培養實驗…………...... 49
陸、結論……………………………………………………………………. 54
柒、參考文獻……………...………………...………………………...…… 66

圖目錄
圖1 三種幾丁質水解酵素水解反應的形式 (1) endochitinase (2) exochitinase
(3) N-acetyl-β-D-hexosaminidase………………………………………. 4
圖2 真菌細胞壁之主要組成示意圖……………...………………………… 6
圖3 真菌細胞壁成份之生合成……………………………………………... 7
圖4 在真菌生長時,HexNAc’ase作為中間介質 (mediator),協同性控制細胞
壁幾丁質水解與合成之生化模型……………….……………..……… 9
圖5 木黴菌與宿主接觸前刻 (pre-contact events) 之超寄生交互作用.… 17
圖6 農桿菌媒介真菌轉形原理示意圖……………………….…………… 22
圖7 藉由 (a) gene disruption 或 (b) gene replacement進行基因轉形…… 27
圖8 Inverse PCR 操作流程示意圖………………………………………… 39
圖9 以 nested PCR,擴增 exc1 之 ORF 兩側外 1.5 kb 之基因片段… 40
圖10 以 nested PCR,擴增 exc2 之 ORF 兩側外 1.5kb 之基因片段.. 41
圖11 質體 pPK2…………………………………………………………… 41
圖12 質體 pKO-exc1……………………………………………………… 42
圖13 質體 pKO-exc2……………………………………………………… 42
圖14 (a) exc1 與 (b) exc2 基因剔除確認示意圖………………………… 45
圖15 利用引子 THexe1F/THexe1R及THexe2F/THexe2R 進行 exc1 & exc2
之 PCR 擴增………………………………………………………….. 55
圖16 對 exc1 之 ORF (a) 5’ 端與 (b) 3’ 端 800 bp 序列進行 RE 切位分
析………………………………………………………………………. 56
圖17 對 exc2 之 ORF (a) 5’ 端與 (b) 3’ 端 700bp 序列進行 RE 切位分
析………………………………………………………………………. 57
圖18 以 PCR 技術擴增 exochitinase 編碼區基因兩側外 1.5 kb 之基因片
段………………………………………………………………………. 58
圖19 以 ATMT 法轉形 T. harzianum…………………………………… 59
圖20 利用引子 THexe1F/THexe1R 對轉形子基因組 DNA 進行 PCR 分
析………………………………………………………………………. 60
圖21 利用引子 THexe1F/THexe1R 對轉形子基因組 DNA 進行 PCR 分
析………………………………………………………………………. 61
圖22 T. harzianum幾丁質外切酵素基因剔除菌株 (△exc1 #12 & △exc2 #06)
固態培養生長情形(a),以及生長曲線圖(b) ………………………... 62
圖23 Gene knockout strain 與植物病原真菌 R. solani 之對峙培養實驗.. 64
圖24 T. harzianum 之 wild type、Δexc1 以及 Δexc2 strains 對 R. solani 進
行 120 hr 對峙培養實驗顯微觀察結果……………………………... 65




附錄目錄
附錄一、本論文所使用之培養基組成……………………………………. 78
附錄二、T. harzianum exochitinase(exc1) cDNA sequence, NCBI……….. 79
附錄三、T. harzianum exochitinase(exc2) cDNA sequence, NCBI………... 81
附錄四、本論文所使用之引子列表………………………………………. 83
附錄五、exc1 之 ORF 完整編碼區定序結果與 cDNA 序列比對結果.. 86
附錄六、exc2 之 ORF 完整編碼區定序結果與 cDNA 序列比對結果.. 90
附錄七、exc1 編碼區與轉譯起始碼 (ATG) 上游 3.5kb 之基因序列… 94
附錄八、exc2 編碼區與轉譯起始碼 (ATG) 上游 3.5kb 之基因序列… 96

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