臺灣博碩士論文加值系統

Trichoderma spp.已被廣泛用於生物防治植物病害。近來研究顯示Trichoderma spp.能分泌一種小分子蛋白質稱之為Small proteins (SM1)。SM1蛋白質具有elicitor作用能誘導植物抗病反應進而達到抗菌。過去研究顯示於棉花植株中發現SM1對於觸發ISR(induced systemic resistance)是不可或缺的，且純化的SM1能有效的於稻米、玉米等植物中表現防禦系統基因或有效的抗病蟲害。本研究目的是基因轉殖菸草表現ThSM1以探討ThSM1是否具有抗病原菌的能力，以瞭解ThSM1是否為生物防治機制之一環。本研究構築pTLPThSM1藉由農桿菌基因轉殖法將其送入菸草；pTLPThSM1在ThSM1之N端接上一段燕麥thaumatin-like protein之signal peptide以利分泌到胞外。經由抗生素篩選和組織培養再生，獲得可能之基因轉殖菸草四株。PCR分析結果顯示，所選取的轉殖植株帶有轉殖基因TLPThSM1。以Real-time RT-PCR分析轉殖植株中有兩株植株會表現，然而強弱不一。利用病原細菌菸草青枯病菌(Ralstonia solanacearum)和病原真菌立枯絲核菌(Rhizoctonia solani)及菌核病菌(Sclerotinia sclerotiorum D5)檢測其抗菌能力，實驗結果顯示能表現轉殖基因TLPThSM1的轉殖植株與未轉殖植株相比並無較明顯的抗菌能力。我們推測這可能是因為轉譯後修飾導致蛋白質與原物種在結構上的差異、啟動子(promoter)未能使轉殖蛋白大量表現於菸草葉片中或SM1蛋白於轉殖植株中的表現為單體或雙體構型上差異所致。

Trichoderma spp. has been widely used as a biocontrol agent. Recent studies showed that Trichoderma spp. produce a small protein (SM1) that acts as an elicitor to trigger the resistance reactions of the colonized plants. Past studies have shown that SM1 is essential to trigger ISR (systemic resistance induced systemic resistance) in cotton, purified SM1 can induce cotton performance of the defense system against the pathogen of Colletotrichum graminicola and SM1 for the defense system to stimulate the corn is very effective. We believe that ThSM1 protein may have the activity of elicitor to trigger resistance reaction of colonized plants against pathogen infection. The purpose of this study is to elucidate the antimicrobial activity of ThSM1 protein in transgenic tobacco. The ThSM1 cDNA was constructed into the pCAMBIA vector and transformed into tobacco by Agrobacterium-mediated transformation. The N-terminus of the ThSM1 was fused to a signal peptide of thaumatin-like protein (TLP). Expression of the transgenes in transgenic tobacco was examined by PCR analysis and Real-time RT-PCR analysis. After antibiotic selection and regeneration, four transgenic tobacco plants were obtained. PCR analysis displayed the existence of transgenic gene TLPThSM1 in all the transgenic tobacco. Real-time PCR analysis of transgenic plants revealed differential expression levels of the transgene in transgenic tobacco. However, the transgenic tobacco plants showed no obvious antimicrobial activity against pathogenic bacterium, Ralstonia solanacearum, or pathogenic fungi, Rhizoctonia solani and Sclerotinia sclerotiorum. We speculated that it might be due to different post-translational modification of the transgenic protein or formation of ThSM1 dimers.

致謝 I
中文摘要 II
英文摘要 V
目錄 1
圖目錄 3
一、研究背景 5
1.1 基因轉殖植物 5
1.2 木黴菌 (Trichoderma spp.) 7
1.2.1 木黴菌介紹 7
1.2.2 木黴菌的分類型態 7
1.2.3 木黴菌的發展史 8
1.2.4 木黴菌的生物防治功能 8
1.3 Small protein 1 (SM1) 10
二、研究目的 15
三、材料與實驗方法 17
3.1 植物材料 17
3.2 農桿菌品系 17
3.3 菸草基因轉殖 17
3.4 植物genomic DNA的抽取 18
3.5 聚合酵素連鎖反應 (Polymerase Chain Reaction, PCR) 20
3.6 菸草葉片總RNA的萃取 20
3.7 總RNA反轉錄成cDNA 21
3.8 即時聚合酶連鎖反應 (Real time PCR) 21
3.9 轉殖菸草對菸草青枯病菌(Ralstonia solanacearum)的抗病能力 22
3.10 轉殖菸草對立枯絲核菌 (Rhizoctonia solani)和菌核病菌 (Sclerotinia sclerotiorum D5)的抗病能力 23
3.11 偵測過氧化氫 (H2O2) 23
四、結果 25
4.1 以農桿菌法轉殖ThSM1基因於菸草 25
4.2 以PCR方法偵測菸草再生植株轉殖基因 26
4.3 以即時定量PCR分析轉殖基因之相對表現量 27
4.4 轉殖菸草抗病原細菌能力 27
4.5 轉殖菸草抗病原真菌能力 28
五、討論 29
六、參考資料 33
圖目錄
圖 一、表現載體pTLPThSM1 39
圖 二、ThSM1基因轉殖菸草生長過程 40
圖 三、PCR檢測轉殖菸草之轉殖基因 41
圖 四、Real-time PCR檢測轉殖菸草內ThSM1轉殖基因表現量 42
圖五、轉殖菸草TM1 – TM4抗Ralstonia solanacearum (Pss4)能力 43
圖六、轉殖菸草TM1 – TM4抗立枯絲核菌 (Rhizoctonia solani)病原真菌能力 44
圖七、檢測轉殖菸草TM1 – TM4抗Rhizoctonia solani能力 45
圖八、轉殖菸草TM1 – TM4感染Rhizoctonia solani病原真菌之病症 46
圖九、轉殖菸草TM1 – TM4感染Rhizoctonia solani後H2O2產生情形 47
圖十、轉殖菸草TM1 – TM4抗菌核病菌(Sclerotinia sclerotiorum) D5能力 48
圖十一、轉殖菸草TM1 – TM4抗Sclerotinia sclerotiorum D5能力 49
圖十二、轉殖菸草TM1 – TM4 感染Sclerotinia sclerotiorum D5之病症 50

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