Trichoderma spp.(木黴菌屬)是普遍存在於土壤中的絲狀真菌，可抑制各種植物病原真菌或誘導植物抗病反應的能力，故被廣泛用於生物防治植物病害。研究顯示Trichoderma spp.所分泌的胞外蛋白質，如Small protein 1 (ThSM1)及L-amino acid oxidase (ThLAAO)具有抗菌效果，但其抗病機制仍不清楚。本研究分為二部分。在第一部分研究中我們擬將PR1b-ThSM1基因轉殖於菸草並表現於細胞間隙，以模擬並探究ThSM1進入於植物內是否具有誘導植物抗病的能力及誘導抗性的可能途徑。實驗結果顯示PR1b-ThSM1基因轉殖菸草具有抑制灰黴菌(Botrytis cinerea B134)和菌核病菌(Sclerotinia sclerotiorum D5)生長能力，並誘導ROS相關基因、茉莉酸和乙烯合成基因及PR protein防禦基因的表現。先前實驗室游心喬學姊建構ThLAAO基因轉殖於菸草中(pThLAAO和pPRlb-ThLAAO載體)，並發現ThLAAO基因轉殖株可抑制病原菌生長，然其誘導抗性的可能途徑並不清楚，故在第二部份實驗中擬探究其誘導抗性的途徑。以pThLAAO載體將ThLAAO基因轉殖於菸草表現可誘導ROS代謝相關基因、茉莉酸和乙烯合成基因的表現。然而、以pPR1b-ThLAAO載體將ThLAAO基因轉殖於菸草表現除了可誘導ROS代謝相關基因、茉莉酸和乙烯水楊酸合成基因表現外，也會誘導水楊酸合成基因及其下游PR protein防禦基因的表現。綜合上述實驗結果證明ThSM1及ThLAAO在基因轉殖株菸草內可抑制病原真菌的生長，且其誘導抗性的途徑可能經由Induced systemic resistance (ISR)誘導抗性。

Trichoderma spp., a filamentous fungi, is commonly found in soil. It can inhibit the growth of various plant pathogenic fungi by inducing plant disease resistance. Therefore, it is widely used in biological control of plant diseases. Several lines of evidences have shown that the extracellular small protein 1 (SM1) and L-Amino acid oxidas (ThLAAO) secreted by Trichoderma spp. exhibit antimicrobial effects. The mechanism of disease resistance, however, is still uclear. This study is divided into two parts. First, we transform the ThSM1 gene into tobacco and explore whether it can inhibit the growth of pathogenic fungi by inducing plant disease resistance. The results show that expression of PR1b-ThSM1 in transgenic tobacco inhibits the growth of Botrytis cinerea and Sclerotinia sclerotiorum, and furthermore, induces the production of reactive oxygen species (ROS), and expression of ROS metabolic genes, jasmoic acid (JA) and ethylene (ET) biosynthetic genes, and PR proteins. Secondly, we plane to elucidate the defense mechanism triggered by LAAO. The results reveal that expression of the ThLAAO gene in transgenic tobacco (L2) triggers ROS production and induces ROS metabolic、JA and ET biosynthetic gene expression. In contrast, in addition to ROS production and expression of ROS metabolic、JA and ET biosynthetic genes, expression of the PR1b-ThLAAO in transgenic tobacco (P10) triggers salicylic acid biosynthetic genes and downstream PR protein genes expression. Taken together, the above results demonstrate that expression of ThSM1 and ThLAAO in transgenic tobacco inhibits the growth of pathogenic fungi, and the defense mechanism involves induced systemic resistance (ISR).

中文摘要 I
英文摘要 III
一、前言 1
1.1 木黴菌(Trichoderma spp.) 1
1.1.1 木黴菌介紹 1
1.1.2 木黴菌分類與特性 1
1.1.3 木黴菌的生物防治功能 1
1.2 木黴菌產生具有誘導抗病能力的蛋白質 2
1.2.1 Small Protein 1 (SM1) 3
1.2.2 Trichoderma harzianum L-Amino acid oxidas (ThLAAO) 4
1.3 Trichoderma spp.所誘導的植物抗性 7
二、研究動機與目的 9
三、實驗設計 11
四、材料與實驗方法 13
4.1 植物材料 13
4.2 病原真菌品系 13
4.3 使用的引子與載體 13
4.4表現pPRlb-ThSM1載體的構築 13
4.4.1 PCR增殖出ThSM1和PRlb DNA 14
4.4.2 Overlapping PCR 14
4.4.3 TA-cloning 14
4.4.4以電穿孔(electroporation)方式進行大腸桿菌之轉型作用(Transformation) 15
4.4.5 以PCR分析檢測轉形株(Transformants) 15
4.4.6 質體DNA(plasmid DNA)萃取 16
4.4.7 限制酶(restriction enzyme)切割與酒精沉澱 16
4.4.8 製備dialysis tubing和gel extraction回收DNA 17
4.4.9 phenol/chloroform酒精沉澱 17
4.5農桿菌之轉型作用 17
4.6農桿菌媒介之基因轉殖法(Agrobacterium-mediated transformation) 18
4.7 抽取基因轉殖株菸草genomic DNA 19
4.8 以聚合酵素連鎖反應(Polymerase Chain Reaction, PCR)檢測轉殖基因 20
4.9抽取基因轉殖菸草植株total RNA 20
4.10 total RNA反轉錄成cDNA 21
4.11 以即時聚合酶鏈鎖反應（Real-time Polymerase Chain Reaction，Real-time PCR）檢測轉殖基因及抗病相關基因之表現量 21
4.12基因轉殖菸草對灰黴菌(Botrytis cinerea B134)和菌核病菌(Sclerotinia sclerotiorum D5)病原真菌之抗病能力 22
4.13偵測O2- 22
4.14偵測過氧化氫(H2O2) 23
4.15 H2O2拮抗試驗 23
4.16 LAAO偶聯FITC 24
4.17 LAAO-FITC與根作用 24
4.18 Protoplasts preparation 25
4.19 Protoplasts固定 25
4.20 Immunofluorescence labeling 26
4.21 H2DCF-DA偵測H2O2 26
4.22 LAA-FITC與protoplasts作用 27
4.23 共軛焦顯微鏡觀察 27
4.24 統計分析 27
五、結果 29
5.1 建立基因轉殖菸草表現木黴菌SM1蛋白質及測試其抗病能力 29
5.1.1建構SM1轉殖基因 29
5.1.2以農桿菌法進行基因轉殖 29
5.1.3以聚合酵素連鎖反應(Polymerase Chain Reaction, PCR)檢測轉殖株是否轉入目標基因 30
5.1.4以即時定量聚合酶鏈鎖反應（Real-Time Quantitative Polymerase Chain Reaction，RT-qPCR）檢測轉殖基因菸草的基因表現量 30
5.1.5轉殖基因菸草對灰黴菌(Botrytis cinerea B134)和菌核病菌(Sclerotinia sclerotiorum D5)病原真菌之抗病能力 30
5.1.6偵測SM1基因轉殖菸草誘導產生過氧化物O2-及H2O2 31
5.1.7以RT-qPCR偵測SM1轉殖菸草防禦基因(Defense genes)之表現 31
5.2 基因轉殖菸草表現木黴菌LAAO蛋白質及測試其防禦基因途徑 32
5.2.1 以RT-qPCR檢測轉殖基因菸草內LAAO基因表現量 32
5.2.2偵測基因轉殖菸草誘導產生超氧負離子(superoxide anion, O2-) 33
5.2.3以RT-qPCR偵測LAAO轉殖菸草防禦基因(Defense genes)之表現。 33
六、討論 35
七、總結 41
八、參考文獻 43

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