木黴菌是目前廣泛應用於農業的生物防治劑。其許多的生物防治機制，已被證實與木黴菌所分泌的分子有關係，這些分子大部分具有抗生素活性、誘導植物抗性和促進生長等特性。哈氏木黴菌細胞外蛋白質–L-氨基酸氧化酶 (Th-LAAO)已被純化，其生物化學也被鑑定。在本次調查中進一步鑑定生物學功能。 Th-LAAO可以誘導甘藍抵抗灰黴菌感染，尤其是用Th-LAAO處理甘藍3天後可以將灰黴菌感染面積降低至1％，在許多防禦相關基因測試中，我們發現植物的基因會透過減少H2O2增加抗壞血酸過氧化物酶(APX)的基因表達。先前實驗室研究證明，哈氏木黴透過增加光合作用、蔗糖生產和運輸相關基因表現來促進甘藍生長。Th-LAAO透過促進Rubisco、Rubisco activase 和 ATP synthase以上光合作用相關的基因表達增加2.54, 2.18, 1.41倍以及蔗糖運輸蛋白1（SUC1）7.6倍而在蔗糖運輸蛋白2 (SUC2)、蔗糖運輸蛋白3 (SUC3)和蔗糖磷酸合成酶 (SPS)中沒有表現出非常相似的特性。對於分泌的代謝產物，哈氏木黴的pachybasin, 大黃酚chrysophanol已經顯示出具抗革蘭氏陰性細菌和灰黴病菌的抗生素活性。哈氏木黴能承受這些分子的毒性，為了瞭解其中分子機制，將IC50大黃酚（30 ppm）測試於哈氏木黴菌，在螢光顯微鏡下，大黃酚處理的哈氏木黴菌絲體比對照組產生更多的H2O2。在處理30 ppm大黃酚後，在哈氏木黴菌中reactive oxygen species (ROS)相關的超氧化物歧化酶 (SOD)表達的變化增加43％（1天）和59％（3天）。處理3天後，過氧化物酶(PEX)表達增加0.23mmol / min / mL（35％）。處理3天後過氧化氫酶(CAT)降至3.04mmol / min / mL（26％），但穀胱甘肽-S-轉移酶(GST)表達沒有顯著變化。因此，哈氏木黴增加SOD和PEX以加速菌絲體中ROS的減少，這可能保護細胞膜的完整性相關。同時，透過二維蛋白質電泳分析測定了哈氏木黴細胞內蛋白質的變異。改變的主要蛋白質是Hexagonal protein (HEX1), nitroreductase (NAD), transaldolase (TRAD)。據文獻回顧，HEX1蛋白的功能可以防止細胞膜損傷和細胞質流失，從而增強維持細胞膜的完整性。SHMT在阿拉伯芥（Arabidopsis thaliana）的抗性增加中具有相對功能。這項研究暨一步了解哈氏木黴分子促進甘藍生長、免疫誘導分子機制；同時保護自身免受抗生素對自身的不利影響的部分分子機制。

Trichoderma spp. are widely used biocontrol agents in agriculture. Among many proposed mechanisms, secreted molecules possess antibiotic activities, induction of resistance of and growth promotion of host being most significant. An extracellular protein of Trichoderma harzianum L-amino acid oxidase (Th-LAAO) has been purified and characterized in biochemistry. Its biological functions were further characterized in this investigation. Th-LAAO treated cabbage 3 days can reduce the Botrytis cinerea infection area down to 1%. The role in cabbage resistance against B. cinerea is contributed to, among many tested defense related genes, increment gene expression in ascorbic peroxidase by reducing H2O2. Previous research in this laboratory showed T. harzianum promoted cabbage growth by increasing genes related photosynthesis and sucrose production and transportation. Th-LAAO showed very similar properties by promoting photosynthesis related genes expression in Rubisco, Rubisco activase and ATP synthase to 2.54, 2.18, 1.41-fold, as well as in sucrose transporter 1 (SUC1) 7.6 fold but not in sucrose transporter 2 (SUC2), sucrose transporter 3 (SUC3) and sucrose phosphate synthase(SPS). To the secreted metabolites chrysophanol of T. harzianum, has been shown antibiotic activities against Gram-negative bacteria and B. cinerea. How does T. harzianum endure these secreted molecules toxicity produced by itself? To discern this, IC50 of chrysophanol, 30 ppm, was applied in T. harzianum culture. Chrysophanol treated T. harzianum mycelia generated more H2O2 than the control group under fluorescent microscope. The change of the reactive oxygen species (ROS) related enzymes superoxide dismutase (SOD) expression was up to 43% (1 day) and 59% (3 days) in T. harzianum after the treatment of 30 ppm chrysophanol. The peroxide (PEX) expression was up to 0.23 mmol/min/mL (35%) after 3 days of the treatment. The catalase (CAT) was down to 3.04 mmol/min/mL (26%) after 3 days of the treatment but there was not significant change in glutathione S-transferase (GST) expression. Therefore, T. harzianum increased the SOD and PEX enzymes to accelerate ROS diminishing in the mycelia that might protect cell membrane integrity. Meanwhile, variations in T. harzianum intracellular proteins were determined by proteome. The major proteins changed are Hexagonal protein (HEX1), nitroreductase (NAD), transaldolase (TRAD) and serine hydroxymethyl transferase (SHMT). The function of HEX1 protein was reported to prevent the cell membrane damage and cytoplasm lose that might enhance the cell membrane. Moreover, SHMT exhibit relative function in resistance increase in Arabidopsis thaliana. This investigation demonstrated possible mechanisms of T. harzianum secreted molecules to benefit cabbage growth meanwhile to protect itself from detrimental effects.

1. Introduction 9
2. Materials and methods 13
2.1. Chemical reagent 13
2.2. Fungi Cultivation 13
2.3. Chemical Chrysophanol 13
2.4. Chrysophanol IC50 of Trichoderma determination. 13
2.5. Chrysophanol treated T. harzianum ETS 323 for proteome 14
2.5.1 Chrysophanol treated with T. harzianum ETS 323 14
2.5.2 Extraction of cytoplasm protein and the protein assay 14
2.6. Brassica oleracea var. capitate (cabbage) cultivation 16
2.7. Th-LAAO purification 16
2.8. Gel stain 17
2.9. 2D-PAGE for the intracellular protein 18
2.10. In-gel digestion and ESI-QUAD-TOF. 18
2.11. Three ways interaction of Th-LAAO, B. oleracea var. capitate and B. cinerea interaction 19
2.12. B. oleracea var. capitate total RNA preparation 20
2.13. cDNA preparation 20
2.14. Reverse transcription-quantitative real-time polymerase chain (RT-qPCR) 21
2.14.1. The primer design of B. oleracea var. capitate 21
2.14.2. The primer design of T. harzianum ETS 323 23
2.15. Observation of ROS fluorescent stain to mycelia 23
2.16. ROS activity 24
2.17. Statistical Analysis 27
3. Results 29
Part I 29
3.1. Th-LAAO enhance cabbage disease resistance. 29
3.2. The Photosynthesis-related genes expression of cabbage increase by treating Th-LAAO 29
3.3. Th-LAAO stimulates the expression of sucrose transport-related genes 30
3.4. Cabbage defense related genes expression in three way interaction 30
Part II 31
3.5. IC50 of Chrysophanol to T. harzianum ETS 323 31
3.6. T. harzianum ETS 323 morphologic change by chrysophanol treatment 31
3.7. Proteome investigation of chrysophanol treated T. harzianum ETS 323 32
3.8. Gene expression of chrysophanol treated T. harzianum ETS 323 32
3.9. ROS H2O2 expression of Chrysophanol treated T. harzianum ETS 323 32
3.10. The enzyme assay of chrysophanol treated T. harzianum ETS 323 ROS 33
4. Discussion 35
5. Conclusion 39
6. Reference 41
7. Figure and result 45
8. Appendix 61

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