臺灣博碩士論文加值系統

植物會產生不同的揮發性有機物(volatile organic compound, VOC)，大部分揮發性化合物與植物的二次代謝物有關，如萜類不只是組成花香的主要成分，還具有其他生理功能，像是吸引昆蟲傳播授粉，直接或間接抗微生物和抗蟲害的植物防禦功能。部分揮發性化合物的生合成途徑中需要O-methyltransferases (OMTs)的參與，OMTs是一個龐大的酵素家族，實驗證實OMTs會參與葡萄酒氣味合成和玫瑰花瓣香味的生合成路徑，也會催化S-adenosylmethionine (SAM)的甲基轉移到二次代謝物或其他小分子的氧上，如類黃酮、生物鹼、植物抗毒素及木質素前驅物等。先前實驗室研究發現植物菌質體Candidatus phytoplasma mali所分泌的致病性蛋白分子SAP11 (secreted AY-WB proteins 11)會抑制菸草揮發性化合物 3-isobutyl-2-methoxypyrazine (IBMP)的產生，IBMP是菸草的主要揮發性氣味分子，會藉由NbOMT1將SAM的甲基團轉移至IBHP，進而形成具揮發性的IBMP分子。透過生物資訊學的分析，發現菸草中還有兩個類似於NbOMT1的基因存在，分別命名為NbOMT2和NbOMT3。目前推測NbOMT3會彌補NbOMT1的功能，但NbOMT3是否也參與IBMP的生合成仍然未知。因此本篇研究使用大腸桿菌系統表現NbOMT3之重組蛋白，發現菌液能夠產生揮發性IBMP分子，將NbOMT3進行酵素活性反應，進一步利用氣相層析質譜分析，證實NbOMT3確實具有甲基轉移酶的酵素活性且可以參與IBMP的生合成。除此之外，利用qRT-PCR分析野生型和35S::SAP11CaPM轉殖菸草中其他OMTs之差異性，發現NbOMT8、NbOMT9、NbOMT10和NbOMT11的基因表現差異最多，此結果顯示SAP11致病性蛋白分子在菸草中也會影響其他OMTs的功能性。

Plants synthesize diversity of volatile organic compounds that attracting pollinators and seed dispersers to protecting themselves from pathogens, parasites and herbivores. We previously reported that transgenic Nicotiana Benthamiana line expressing the secreted effector SAP11 from Candidatus Phytoplasma mali can suppress the biosynthesis of 3-isobutyl-2-methoxypyrazine (IBMP), a volatile organic compound. We further showed that NbOMT1 is the key enzyme, which catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to 3-isobutyl-2-hydroxypyrazine (IBHP) to produce IBMP. However, two uncharacterized O-methyltransferases, NbOMT2 and NbOMT3, which share high similarity with NbOMT1 are found in the genome of N.benthamiana. In this study, the recombinant protein was produced and the O-methyltransferase activity of NbOMT3 was tested in vivo and in vitro using HS-SPME coupled GC-MS. We showed that NbOMT3 can catalyze the production of IBMP in vivo and in vitro. Using qRT-PCR analysis, we further showed that the transcript levels of NbOMT8, NbOMT9, NbOMT10 and NbOMT11 were also affected by SAP11CaPM. According to all result, we suggest that SAP11 may interfere the production of secondary metabolitites in N.benthamiana.

第一章　前言 1
一、O-methyltransferase之介紹 1
二、IBMP揮發性分子 2
三、植物菌質體(Phytoplasma)之介紹 2
四、植物菌質體命名與分群 3
五、植物菌質體分泌系統 4
第二章　研究目的與策略 6
第三章　材料與方法 7
※材料 7
※方法 7
一、載體構築 7
二、農桿菌轉型株之建立 10
三、酵母菌轉型株之建立 10
四、西方點墨法(Western blotting) 11
五、圓葉菸草RNA萃取 12
六、反轉錄酶反應 (reverse transcription) 13
七、即時定量聚合酶連鎖反應(real-time PCR) 13
八、固相微萃取法(Solid-phase microextraction, SPME) 13
九、酵母菌破菌 14
十、表現及純化重組蛋白 15
十一、酵素活性反應 16
第四章　結果 17
一、NbOMT3重組蛋白之甲基轉移酶酵素活性的in vivo分析 17
二、NbOMT3重組蛋白之甲基轉移酶酵素活性的in vitro分析 17
三、利用酵母系統表現NbOMT1並檢測其氣味 18
四、利用表現NbOMT1-SFP的酵母菌進行麵包烘焙味 19
五、菸草Methyltransferase之親緣性分析 19
六、 SAP11CaPM 轉基因植物Group II NbOMTs相關基因之表現量分析 20
第五章　討論 21
第六章　圖、表 23
第七章　附錄 32
第八章　參考文獻 47

Bai, X., Correa, V.R., Toruño, T.Y., Ammar, E.-D., Kamoun, S., and Hogenhout, S.A. (2008). AY-WB Phytoplasma Secretes a Protein That Targets Plant Cell Nuclei. Molecular Plant-Microbe Interactions 22, 18-30.
Bai, X., Zhang, J., Ewing, A., Miller, S.A., Jancso Radek, A., Shevchenko, D.V., Tsukerman, K., Walunas, T., Lapidus, A., Campbell, J.W., and Hogenhout, S.A. (2006b). Living with Genome Instability: the Adaptation of Phytoplasmas to Diverse Environments of Their Insect and Plant Hosts. Journal of bacteriology 188, 3682-3696.
Bertaccini, A., and Duduk, B. (2009). Phytoplasma and phytoplasma diseases: a review of recent research.
Bertaccini, A., Duduk, B., Paltrinieri, S., and Contaldo, N. (2014). Phytoplasmas and Phytoplasma Diseases: A Severe Threat to Agriculture. American Journal of Plant Sciences 05, 1763-1788.
Dunlevy, J.D., Dennis, E.G., Soole, K.L., Perkins, M.V., Davies, C., and Boss, P.K. (2013). A methyltransferase essential for the methoxypyrazine-derived flavour of wine. The Plant Journal 75, 606-617.
Gang, D.R. (2005). EVOLUTION OF FLAVORS AND SCENTS. Annual Review of Plant Biology 56, 301-325.
Guillaumie, S., Ilg, A., Réty, S., Brette, M., Trossat-Magnin, C., Decroocq, S., Léon, C., Keime, C., Ye, T., Baltenweck-Guyot, R., Claudel, P., Bordenave, L., Vanbrabant, S., Duchêne, E., Delrot, S., Darriet, P., Hugueney, P., and Gomès, E. (2013). Genetic Analysis of the Biosynthesis of 2-Methoxy-3-Isobutylpyrazine, a Major Grape-Derived Aroma Compound Impacting Wine Quality. Plant physiology 162, 604-615.
Hogenhout, S.A., Oshima, K., Ammar, E.-D., Kakizawa, S., Kingdom, H.N., and Namba, S. (2008). Phytoplasmas: bacteria that manipulate plants and insects. Molecular plant pathology 9, 403-423.
Humphreys, J.M., and Chapple, C. (2002). Rewriting the lignin roadmap. Current Opinion in Plant Biology 5, 224-229.
Joshi, C., and Chiang, V. (1998). Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases. Plant Mol Biol 37, 663-674.
Kamińska, M., and Korbin, M. (1999). Graft and dodder transmission of phytoplasma affecting lily to experimental hosts. Acta Physiol Plant 21, 21-26.
Kavousi, A., Chi, H., Talebi, K., Bandani, A., Ashouri, A., and Hosseini Naveh, V. (2009). Demographic Traits of Tetranychus urticae (Acari: Tetranychidae) on Leaf Discs and Whole Leaves. Journal of Economic Entomology 102, 595-601.
Knudsen, J., Eriksson, R., Gershenzon, J., and Ståhl, B. (2006). Diversity and distribution of floral scent. Bot. Rev 72, 1-120.
Lam, K.C., Ibrahim, R.K., Behdad, B., and Dayanandan, S. (2007). Structure, function, and evolution of plant O-methyltransferases. Genome 50, 1001-1013.
Lee, I.-M., Gundersen-Rindal, D.E., and Bertaccini, A. (1998). Phytoplasma: Ecology and Genomic Diversity. Phytopathology 88, 1359-1366.
Lee, I.-M., Davis, R.E., and Gundersen-Rindal, D.E. (2000). Phytoplasma: Phytopathogenic Mollicutes. Annual Review of Microbiology 54, 221-255.
Lee, I.M., Bottner, K.D., Secor, G., and Rivera-Varas, V. (2006). "Candidatus Phytoplasma americanum", a phytoplasma associated with a potato purple top wilt disease complex. International journal of systematic and evolutionary microbiology 56, 1593-1597.
Lu, Y.-T., Li, M.-Y., Cheng, K.-T., Tan, C.M., Su, L.-W., Lin, W.-Y., Shih, H.-T., Chiou, T.-J., and Yang, J.-Y. (2014). Transgenic Plants That Express the Phytoplasma Effector SAP11 Show Altered Phosphate Starvation and Defense Responses. Plant physiology 164, 1456-1469.
Paré, P.W., and Tumlinson, J.H. (1999). Plant Volatiles as a Defense against Insect Herbivores. Plant physiology 121, 325-332.
Rimando, A.M., Pan, Z., Polashock, J.J., Dayan, F.E., Mizuno, C.S., Snook, M.E., Liu, C.-J., and Baerson, S.R. (2012). In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression. Plant Biotechnology Journal 10, 269-283.
Scalliet, G., Piola, F., Douady, C.J., Réty, S., Raymond, O., Baudino, S., Bordji, K., Bendahmane, M., Dumas, C., Cock, J.M., and Hugueney, P. (2008). Scent evolution in Chinese roses. Proceedings of the National Academy of Sciences 105, 5927-5932.
Schilmiller, A.L., Last, R.L., and Pichersky, E. (2008). Harnessing plant trichome biochemistry for the production of useful compounds. The Plant Journal 54, 702-711.
Schmidlin, L., Poutaraud, A., Claudel, P., Mestre, P., Prado, E., Santos-Rosa, M., Wiedemann-Merdinoglu, S., Karst, F., Merdinoglu, D., and Hugueney, P. (2008). A Stress-Inducible Resveratrol O-Methyltransferase Involved in the Biosynthesis of Pterostilbene in Grapevine. Plant physiology 148, 1630-1639.
Schmidt, A., Li, C., Shi, F., Jones, A.D., and Pichersky, E. (2011). Polymethylated Myricetin in Trichomes of the Wild Tomato Species Solanum habrochaites and Characterization of Trichome-Specific 3′/5′- and 7/4′-Myricetin O-Methyltransferases. Plant physiology 155, 1999-2009.
Seemuller, E. (2004). ''Candidatus Phytoplasma mali'', ''Candidatus Phytoplasma pyri'' and ''Candidatus Phytoplasma prunorum'', the causal agents of apple proliferation, pear decline and European stone fruit yellows, respectively. International journal of systematic and evolutionary microbiology 54, 1217-1226.
Siddall, E.C., and Marples, N.M. (2008). Better to be bimodal: the interaction of color and odor on learning and memory. Behavioral Ecology 19, 425-432.
Sugio, A., and Hogenhout, S.A. (2012). The genome biology of phytoplasma: modulators of plants and insects. Current Opinion in Microbiology 15, 247-254.
Sugio, A., MacLean, A.M., and Hogenhout, S.A. (2014). The small phytoplasma virulence effector SAP11 contains distinct domains required for nuclear targeting and CIN-TCP binding and destabilization. New Phytologist 202, 838-848.
Sugio, A., Kingdom, H.N., MacLean, A.M., Grieve, V.M., and Hogenhout, S.A. (2011a). Phytoplasma protein effector SAP11 enhances insect vector reproduction by manipulating plant development and defense hormone biosynthesis. Proceedings of the National Academy of Sciences 108, E1254–E1263.
Sugio, A., MacLean, A.M., Kingdom, H.N., Grieve, V.M., Manimekalai, R., and Hogenhout, S.A. (2011b). Diverse targets of phytoplasma effectors: from plant development to defense against insects. Annual review of phytopathology 49, 175-195.
Wheeler, C., and Cardé, R. (2013). Defensive Allomones Function as Aggregation Pheromones in Diapausing Ladybird Beetles, Hippodamia convergens. J Chem Ecol 39, 723-732.