臺灣博碩士論文加值系統

木黴菌是很重要的生物防治微生物，對於許多植物病原真菌都具有防治效果。大多數的木黴菌皆可藉由分泌一些溶酶來達到此效果，例如:L型胺基酸氧化酶、β-1,3-葡萄糖酶和幾丁質分解酵素。灰黴菌是很普遍的植物病原真菌，它會造成大部分的植物損傷，會使蔬菜、水果和花卉的產量減少。在我們之前的研究中發現當哈氏木黴菌存在於去活化的灰黴菌培養液裡會分泌出L型胺基酸氧化酶，而且此酵素具有拮抗病原真菌的作用。這裡，我們提出假說:哈氏木黴菌拮抗灰黴菌的機制屬於兩步驟的拮抗；初期和後期的菌絲纏繞速率是不同的。在我們的研究中發現，由哈氏木黴菌所分泌出的L型胺基酸氧化酶可以有效的抑制灰黴菌菌絲生長，並且可以造成灰黴菌菌絲內細胞質產生空泡，進而造成菌絲溶解。在實體植物實驗當中，可以發現哈氏木黴菌L型胺基酸氧化酶可以在有灰黴菌的存在底下保護菸草葉與蘋果的完整性，致使灰黴菌不傷害植物。而進一步的觀察到類似細胞凋亡的現象出現在哈氏木黴菌L型胺基酸氧化酶處理灰黴菌菌絲之後，例如:自由基的形成，此建議哈氏木黴菌L型胺基酸氧化酶造成灰黴菌產生自由基而形成一系列的細胞凋亡現象。我們利用西方墨點法之紀錄，確認灰黴菌的細胞凋亡途徑。在結果發現，當灰黴菌在含有哈氏木黴菌L型胺基酸氧化酶的存在可偵測到細胞凋亡因子，例如:細胞質偵測到細胞色素c、蛋白酶3和蛋白酶9。而後再用二維電泳方式深入探討哈氏木黴菌L型胺基酸氧化酶存在灰黴菌實驗中時，灰黴菌蛋白分子層面之變化。我們鑑定出許多分泌的胞外蛋白，包含:轉譯蛋白、核醣體蛋白、摺疊蛋白用的伴隨蛋白、能量蛋白及訊號傳遞蛋白。此實驗偵測到對於抗氧化很有效的超氧化歧化酶存在於未處理哈氏木黴菌L型胺基酸氧化酶的灰黴菌菌絲中，這可能是哈氏木黴菌L型胺基酸氧化酶拮抗灰黴菌很重要的因素之一，加入L型胺基酸氧化酶的灰黴菌則因要自我保護及抵抗木黴菌L型胺基酸氧化酶過度表達能量蛋白、轉譯蛋白及分子摺疊蛋白而造成一系列不正常反應現象。隨後，我們利用克隆的方式將新型哈氏木黴菌L型胺基酸氧化酶藉由大腸桿菌過表達的系統表達出來後再做純化，我們將哈氏木黴菌L型胺基酸氧化酶與其他六種物種的L型胺基酸氧化酶做序列比對，序列相似度有9%-24%，分子量為52千道爾頓，表達的哈氏木黴菌L型胺基酸氧化酶的最佳胺基酸受質是左型苯丙氨酸，並且在酸鹼值7以及溫度40 °C時活性最好。在圓二色光譜顯示過表達出的哈氏木黴菌L型胺基酸氧化酶的二級結構比例為17% α-螺旋， 28% β-折板， 和 55% 不規則形; 此一蛋白並具有抗革蘭氏陰性菌及革蘭氏陽性菌的活性。我們也提出哈氏木黴菌L型胺基酸氧化酶的三維立體預測結構圖，可以提供更多關於過表達的哈氏木黴菌L型胺基酸氧化酶可能是利用N端序列來達到抗菌的資訊。以上一系列關於哈氏木黴菌L型胺基酸氧化酶一系列實驗，可以幫助我們更了解哈氏木黴菌L型胺基酸氧化酶整個拮抗真菌及抗細菌的詳細作用機制。

Trichoderma harzianum is an important biocontrol agent of several important plant pathogenic fungi. Trichoderma species attacks other fungi by secreting lytic enzymes, including L-amino acid oxidase, β-1,3-glucanase and chitinolytic enzymes. Botrytis cinerea is a common pathogenic fungus which causes blight in most ornamental plants. It may be responsible for serious diseases and postharvest losses in fruits, vegetables and flowers. Previous studies have shown that the extracellular proteins of T. harzianum ETS 323 grown in the presence of deactivated B. cinerea in culture include a putative L-amino acid oxidase and have suggested the involvement of this enzyme in the antagonistic mechanism. Here, we hypothesized that the mycoparasitic process of Trichoderma spp. against B. cinerea involves 2 steps, that is, an initial hyphal coiling stage and a subsequent hyphal coiling stage, with different coiling rates. In addition, an L-amino acid oxidase (Th-L-AAO) was identified from T. harzianum ETS 323. The secretion of Th-L-AAO was increased when T. harzianum ETS 323 was grown with deactivated hyphae of B. cinerea. Moreover, in vitro assays indicated that Th-L-AAO effectively inhibited B. cinerea hyphal growth, caused cytosolic vacuolization in the hyphae, and led to hyphal lysis. Th-L-AAO also showed disease control against the development of B. cinerea on postharvest apple fruits and tobacco leaves. An apoptosis-like response, including the generation of reactive oxygen species, was observed in B. cinerea after treatment with Th-L-AAO, suggesting that Th-L-AAO triggers programmed cell death in B. cinerea. We show that release of the apoptotic factors cytochrome c, caspase 3, and caspase 9, upon treatment with Trichoderma harzianum-derived L-amino acid oxidase by western blot. Furthermore, we report on a proteomic analysis of secreted proteins from B. cinerea during treatment with T. harzianum ETS 323 L-phenylalanine oxidase (Th-L-AAO) using two-dimensional electrophoresis (2DE). We identified numerous extracellular proteins in the secretions, including translation proteins, ribosomal proteins, and chaperone proteins for folding, metabolism, energy production, and signal transduction. We detected superoxide dismutase (SOD) in un-treated B. cinerea. The results of this study provide important insights into the mechanism of Th-L-AAO action against B. cinerea. In cloning study, we report of the cloning of cDNA encoding a novel T. harzianum ETS 323 L-amino acid oxidase. The protein was overexpressed in Escherichia coli and purified to homogeneity. Comparisons of its deduced amino acid sequence with the sequence of other L-AAOs revealed the similarity to be between 9% and 24%. The molecular mass of the purified protein was 52 kDa, as determined by SDS-PAGE. Th-L-AAO showed detectable optimal activity substrate on phenylalanine. The enzyme optimal pH and temperature for activity were 7 and 40°C, respectively. In Circular Dichroism spectroscopy indicated that the secondary structure of Th-L-AAO is composed of 17% α-helices, 28% β-sheets, and 55% random coils. The bacterially expressed Th-L-AAO also mediated antibacterial activity against both gram-positive and gram-negative food spoilage organisms. A 3D protein structure was created to provide more information about the structural composition of Th-L-AAO, suggesting that the N-terminal sequence of Th-L-AAO may have contributed to the antibacterial activity of this protein.

誌謝 -----------------------------------------------------I
中文摘要 -------------------------------------------------II
Abstract ------------------------------------------------IV
Table of Contents ------------------------------------- VII
Introduction -------------------------------------------- 1
Materials and Methods ----------------------------------- 6
Results ------------------------------------------------ 27
Discussion --------------------------------------------- 45
Conclusion --------------------------------------------- 54
Tables ------------------------------------------------- 56
Figures ------------------------------------------------ 67
References --------------------------------------------- 97
Publications ------------------------------------------ 113

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