病菌或有益微生物誘發植物抗性主要是經由誘導植物產生內源性分子進而誘發植物抗病反應而成。Trichoderma harzianum產生的L-amino acid oxidase (ThLAAO)對植物病原真菌如Botrytis cinerea及Rhizoctonia solani產生抗病，且可誘發H2O2產生及誘導植物賀爾蒙(如水楊酸、乙烯、茉莉酸)生合成基因及防禦基因表現。此外、ThLAAO蛋白質可從植物根部吸收進入維管束，並且可運輸至葉肉細胞內之葉綠體。故推論T. harzianum可產生外源性分子(如ThLAAO)從植物根部進入，然後經維管束運輸至葉子誘發植物抗性。本研究在ThLAAO基因後面接上螢光蛋白基因如green fluorescence protein (GFP)及Orange fluorescence protein (OFP)，並以T. harzianum LAAO gene、enolase gene及Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase gene的promoters驅動ThLAAO表現，透過基因轉形建構木黴菌LAAO-GFP及LAAO-OFP轉形株。我們將檢測這些轉形株是否會表現LAAO-GFP及LAAO-OFP基因及蛋白質。日後將藉此觀測木黴菌所分泌的LAAO-GFP或LAAO-OFP是否可以運輸至植物的各個組織。

The mechanisms for activating disease resistance are mainly induced by endogenous molecules produced by plants. L-amino acid oxidase (ThLAAO) produced by Trichoderma harzianum has an antagonistic effect on plant pathogenic fungi such as Botrytis cinerea and Rhizoctonia solani, and can induce the production of H2O2 and plant hormones (such as salicylic acid, ethylene, jasmonic acid) biosynthesis gene and defense gene expression. In addition, ThLAAO can be absorbed and transported from plant roots to chloroplasts in mesophyll cells. Therefore, it is inferred that Trichoderma spp. can produce exogenous molecules, e.g., ThLAAO, that can directly enter the vascular system of plants and be transported to various parts to induce disease resistance in plants. In this study, the green fluorescence protein (GFP) and orange fluorescent protein (OFP) was cloned into the 3’-end of ThLAAO gene and triggered by the promoters of ThLAAO, enolase gene or Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase gene. These constructs were transformed into Trichoderma via Agrobacterium-mediated transformation. We will detect the expression of LAAOA-GFP and LAAO-OFP, and the Trichoderma transformants will be used to determine whether the LAAOA-GFP and LAAO-OFP secreted by Trichoderma in the soil can be transported to various tissues in plants.

中文摘要: I
英文摘要: II
目錄: III
一、 前言 1
1. 木黴菌及其生物防治功能 1
2. 植物系統性抗性 2
3. 木黴菌誘導系統性抗性 3
4. 木黴菌胞外蛋白質—左旋胺基酸氧化酶（L-amino acid oxidase） 4
5. 葉綠體在植物免疫之角色 5
二、 研究目的 6
三、 實驗設計 7
四、 材料方法 8
1. 菌種 8
2. 引子及質體 8
3. 質體DNA的抽取 8
4. TA cloning 9
5. 製作大腸桿菌勝任細胞及電穿孔 10
6. 快速篩檢確認轉型株 11
7. 製作農桿菌AGL1勝任細胞及電穿孔 11
8. 農桿菌法轉形木黴菌 12
9. 構築Plaao-LAAO-GFP表現載體 13
10. 構築Plaao-Peno-LAAO-OFP-Teno-Tlaao表現載體 14
11. 構築Peno-LAAO-GFP-Teno表現載體 17
12. 構築Pgpd-LAAO-GFP-Ttrp表現載體 18
13. 抽取木黴菌轉殖株基因 21
14. 抽取木黴菌mRNA並反轉錄至cDNA 22
15. 木黴菌胞外蛋白質抽取及偵測 23
16. 以共軛焦顯微鏡偵測轉殖株螢光蛋白表現 24
五、 結果與討論 25
1. 建構LAAO-GFP及LAAO-OFP轉殖基因 25
2. 檢測木黴菌轉形株中之LAAO-GFP及LAAO-OFP轉基因 27
3. 檢測木黴菌轉形株中轉基因表現 30
4. 檢測木黴菌轉形株表現LAAO-GFP及LAAO-OFP胞外蛋白 31
5. 以共軛焦顯微鏡偵測木黴菌轉形株螢光蛋白表現 32
六、 總結 33
七、 參考文獻 34

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