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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 陳昭瑩 | |
dc.contributor.author | Tzu-Chun Lin | en |
dc.contributor.author | 林姿均 | zh_TW |
dc.date.accessioned | 2021-06-17T08:42:10Z | - |
dc.date.available | 2021-08-13 | |
dc.date.copyright | 2019-08-13 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-07 | |
dc.identifier.citation | 1. 潘映潔。2015。LsGRP1C 誘導百合灰黴病菌之程序性細胞死亡現象。台灣大學植物病理與微生物學系碩士論文。
2. 劉芳瑋。2016。抗菌蛋白 LsGRP1 之 N 端區段對百合灰黴病之孢子發芽促進作用。台灣大學植物病理與微生物學系碩士論文。 3. 施侑廷。2018。百合防禦相關蛋白 LsGRP1 誘導植物抗灰黴病菌之應用。台灣大學植物病理與微生物學系碩士論文。 4. Baum, J. A., Bogaert, T., Clinton, W., Heck, G. R., Feldmann, P., Ilagan, O., Johnson, S., Plaetinck, G., Munyikwa, T., and Pleau, M. 2007. Control of coleopteran insect pests through RNA interference. Nature Biotechnology 25:1322. 5. Blow, D. M., Birktoft, J. J., and Hartley, B. S. 1969. Role of a buried acid group in the mechanism of action of chymotrypsin. Nature 221:337. 6. Bocca, S. N., Magioli, C., Mangeon, A., Junqueira, R. M., Cardeal, V., Margis, R., and Sachetto-Martins, G. 2005. Survey of glycine-rich proteins (GRPs) in the Eucalyptus expressed sequence tag database (ForEST). Genetics Molecular Biology 28:608-624. 7. Boller, T., and He, S. Y. 2009. Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens. Science 324:742-744. 8. Brodersen, P., and Voinnet, O. 2006. The diversity of RNA silencing pathways in plants. Trends in Genetics 22:268-280. 9. Bryan, P. N. 2000. Protein engineering of subtilisin. Biochimica et Biophysica Acta -Protein Structure Molecular Enzymology 1543:203-222. 10. Buscaill, P. 2016. A protease of the subtilase family negatively regulates plant defence through its interaction with the Arabidopsis transcription factor AtMYB30. Institut National Polytechnique de Toulouse. Doctoral dissertation. 11. Castel, S. E., and Martienssen, R. A. 2013. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nature Reviews Genetics 14:100. 12. Cedzich, A., Huttenlocher, F., Kuhn, B. M., Pfannstiel, J., Gabler, L., Stintzi, A., and Schaller, A. 2009. The protease-associated domain and C-terminal extension are required for zymogen processing, sorting within the secretory pathway, and activity of tomato subtilase 3 (SlSBT3). Journal of Biological Chemistry 284:14068-14078. 13. Chalfoun, N. R., Grellet-Bournonville, C. F., Martínez-Zamora, M. G., Díaz-Perales, A., Castagnaro, A. P., and Díaz-Ricci, J. C. 2013. Purification and characterization of AsES protein a subtilisin secreted by Acremonium strictum is a novel plant defense elicitor. Journal of Biological Chemistry 288:14098-14113. 14. Chichkova, N. V., Shaw, J., Galiullina, R. A., Drury, G. E., Tuzhikov, A. I., Kim, S. H., Kalkum, M., Hong, T. B., Gorshkova, E. N., and Torrance, L. 2010. Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity. The EMBO Journal 29:1149-1161. 15. Coffeen, W. C., and Wolpert, T. J. 2004. Purification and characterization of serine proteases that exhibit caspase-like activity and are associated with programmed cell death in Avena sativa. Plant Cell 16:857-873. 16. Crétin, C., and Puigdomènech, P. 1990. Glycine-rich RNA-binding proteins from Sorghum vulgare. Plant Molecular Biology 15:783-785. 17. Davuluri, G. R., Van Tuinen, A., Fraser, P. D., Manfredonia, A., Newman, R., Burgess, D., Brummell, D. A., King, S. R., Palys, J., and Uhlig, J. 2005. Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nature Biotechnology 23:890. 18. Dolgov, S., Mikhaylov, R., Serova, T., Shulga, O., and Firsov, A. 2010. Pathogen-derived methods for improving resistance of transgenic plums (Prunus domestica L.) for Plum pox virus infection. Julius-Kühn-Archiv 427:133. 19. Doss, R. P., Christian, J. K., and Chastagner, G. A. 1988. Infection of Easter lily leaves from conidia of Botrytis elliptica. Canadian Journal of Botany 66:1204-1208. 20. Gómez, J., Sánchez-Martínez, D., Stiefel, V., Rigau, J., Puigdomènech, P., and Pagès, M. 1988. A gene induced by the plant hormone abscisic acid in response to water stress encodes a glycine-rich protein. Nature 334:262. 21. Gottwald, S., Samans, B., Lück, S., and Friedt, W. 2012. Jasmonate and ethylene dependent defence gene expression and suppression of fungal virulence factors: two essential mechanisms of Fusarium head blight resistance in wheat? BMC Genomics 13:369. 22. Hsiang, T., Hsieh, T. F., and Chastagner, G. A. 2001. Relative sensitivity to the fungicides benomyl and iprodione of Botrytis elliptica from Taiwan and the Northwestern USA. Plant Pathology Bulletin 10:93-95. 23. Jones, B., Sun, F., and Marchesi, J. R. 2007. Using skimmed milk agar to functionally screen a gut metagenomic library for proteases may lead to false positives. Letters in Applied Microbiology 45:418-420. 24. Jones, J. D., and Dangl, J. L. 2006. The plant immune system. Nature 444:323. 25. Kabbage, M., Kessens, R., Bartholomay, L. C., and Williams, B. 2017. The life and death of a plant cell. Annual Review of Plant Biology 68:375-404. 26. Keller, B., and Baumgartner, C. 1991. Vascular-specific expression of the bean GRP 1.8 gene is negatively regulated. Plant Cell 3:1051-1061. 27. Keller, B., Sauer, N., and Lamb, C. 1988. Glycine‐rich cell wall proteins in bean: gene structure and association of the protein with the vascular system. The EMBO Journal 7:3625-3633. 28. Ketting, R. F. 2011. The many faces of RNAi. Developmental Cell 20:148-161. 29. Kim, Y.-O., Pan, S., Jung, C.-H., and Kang, H. 2007. A zinc finger-containing glycine-rich RNA-binding protein, atRZ-1a, has a negative impact on seed germination and seedling growth of Arabidopsis thaliana under salt or drought stress conditions. Plant Cell Physiology 48:1170-1181. 30. Koch, A., Biedenkopf, D., Furch, A., Weber, L., Rossbach, O., Abdellatef, E., Linicus, L., Johannsmeier, J., Jelonek, L., and Goesmann, A. 2016. An RNAi-based control of Fusarium graminearum infections through spraying of long dsRNAs involves a plant passage and is controlled by the fungal silencing machinery. PLoS Pathogens 12:e1005901. 31. Koch, A., and Kogel, K. H. 2014. New wind in the sails: improving the agronomic value of crop plants through RNA i‐mediated gene silencing. Plant Biotechnology Journal 12:821-831. 32. Koch, A., Kumar, N., Weber, L., Keller, H., Imani, J., and Kogel, K.-H. 2013. Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species. Proceedings of the National Academy of Sciences, USA 110:19324-19329. 33. Lei, M., and Wu, R. 1991. A novel glycine-rich cell wall protein gene in rice. Plant Molecular Biology 16:187-198. 34. Levin, E., Kishore, A., Ballester, A. R., Raphael, G., Feigenberg, O., Liu, Y., Norelli, J., Gonzalez-Candelas, L., Wisniewski, M., and Droby, S. 2019. Identification of pathogenicity-related genes and the role of a subtilisin-related peptidase S8 (PePRT) in authophagy and virulence of Penicillium expansum on apples. Postharvest Biology Technology 149:209-220. 35. Lilley, C., Davies, L., and Urwin, P. 2012. RNA interference in plant parasitic nematodes: a summary of the current status. Parasitology 139:630-640. 36. Lin, C.-H., Chang, M.-W., and Chen, C.-Y. 2014. A potent antimicrobial peptide derived from the protein LsGRP1 of Lilium. Phytopathology 104:340-346. 37. Lin, C.-H., and Chen, C.-Y. 2014. Characterization of the dual subcellular localization of Lilium LsGRP1, a plant class II glycine-rich protein. Phytopathology 104:1012-1020. 38. Linderstrøm-Lang, K., and Ottesen, M. 1947. A new protein from ovalbumin. Nature 159:807. 39. Liu, Q., and Paroo, Z. 2010. Biochemical principles of small RNA pathways. Annual Review of Biochemistry 79:295-319. 40. Liu, Y.-H., Huang, C.-J., and Chen, C.-Y. 2008. Evidence of induced systemic resistance against Botrytis elliptica in lily. Phytopathology 98:830-836. 41. Lu, Y.-Y., and Chen, C.-Y. 2005. Molecular analysis of lily leaves in response to salicylic acid effective towards protection against Botrytis elliptica. Plant Science 169:1-9. 42. Lu, Y.-Y., Liu, Y.-H., and Chen, C.-Y. 2007. Stomatal closure, callose deposition, and increase of LsGRP1-corresponding transcript in probenazole-induced resistance against Botrytis elliptica in lily. Plant Science 172:913-919. 43. Mangeon, A., Junqueira, R. M., and Sachetto-Martins, G. 2010. Functional diversity of the plant glycine-rich proteins superfamily. Plant Signaling Behavior 5:99-104. 44. Matsubara, H., Kasper, C. B., Brown, D. M., and Smith, E. L. 1965. Subtilisin BPN'I. Physical properties and amino acid composition. Journal of Biological Chemistry 240:1125-1130. 45. Matthews, V. 2007. The international lily register and checklist, 2007. Fourth Edition. London: Royal Horticultural Society. 46. Mayfield, J. A., and Preuss, D. 2000. Rapid initiation of Arabidopsis pollination requires the oleosin-domain protein GRP17. Nature Cell Biology 2:128. 47. Mousavi, A., and Hotta, Y. 2005. Glycine-rich proteins. Applied Biochemistry Biotechnology 120:169-174. 48. Mundy, J., and Chua, N.-H. 1988. Abscisic acid and water‐stress induce the expression of a novel rice gene. The EMBO Journal 7:2279-2286. 49. Muszewska, A., Taylor, J. W., Szczesny, P., and Grynberg, M. 2011. Independent subtilases expansions in fungi associated with animals. Molecular Biology Evolution 28:3395-3404. 50. Nowara, D., Gay, A., Lacomme, C., Shaw, J., Ridout, C., Douchkov, D., Hensel, G., Kumlehn, J., and Schweizer, P. 2010. HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22:3130-3141. 51. Nunes, C. C., and Dean, R. A. 2012. Host‐induced gene silencing: a tool for understanding fungal host interaction and for developing novel disease control strategies. Molecular Plant Pathology 13:519-529. 52. Olivieri, F., Godoy, A. V., Escande, A., and Casalongué, C. A. 1998. Analysis of intercellular washing fluids of potato tubers and detection of increased proteolytic activity upon fungal infection. Physiologia Plantarum 104:232-238. 53. Olivieri, F., Zanetti, M. E., Oliva, C. R., Covarrubias, A. A., and Casalongué, C. A. 2002. Characterization of an extracellular serine protease of Fusarium eumartii and its action on pathogenesis related proteins. European Journal of Plant Pathology 108:63-72. 54. Page, M. J., and Di Cera, E. 2008. Serine peptidases: classification, structure and function. Cellular Molecular Life Sciences 65:1220-1236. 55. Park, C. J., Park, C. B., Hong, S.-S., Lee, H.-S., Lee, S. Y., and Kim, S. C. 2000. Characterization and cDNA cloning of two glycine-and histidine-rich antimicrobial peptides from the roots of shepherd's purse, Capsella bursa-pastoris. Plant Molecular Biology 44:187-197. 56. Pearce, G., Yamaguchi, Y., Barona, G., and Ryan, C. A. 2010. A subtilisin-like protein from soybean contains an embedded, cryptic signal that activates defense-related genes. Proceedings of the National Academy of Sciences, USA 107:14921-14925. 57. Plattner, S., Gruber, C., Altmann, F., and Bohlmann, H. 2014. Self-processing of a barley subtilase expressed in E. coli. Protein Expressionand Purification 101:76-83. 58. Rawlings, N. D., and Barrett, A. J. 1993. Evolutionary families of peptidases. Biochemical Journal 290:205-218. 59. Rawlings, N. D., and Barrett, A. J. 1994. Families of serine peptidases. Pages 19-61 in: Methods in Enzymology, vol. 244. Elsevier. 60. Rohde, W., Rosch, K., Kröger, K., and Salamini, F. 1990. Nucleotide sequence of aHordeum vulgare gene encoding a glycine-rich protein with homology to vertebrate cytokeratins. Plant Molecular Biology 14:1057-1059. 61. Sachetto-Martins, G., Franco, L. O., and de Oliveira, D. E. 2000. Plant glycine-rich proteins: a family or just proteins with a common motif? Biochimica et Biophysica Acta - Gene Structure Expression 1492:1-14. 62. Saitoh, H., Fujisawa, S., Ito, A., Mitsuoka, C., Berberich, T., Tosa, Y., Asakura, M., Takano, Y., and Terauchi, R. 2009. SPM1 encoding a vacuole-localized protease is required for infection-related autophagy of the rice blast fungus Magnaporthe oryzae. FEMS Microbiology Letters 300:115-121. 63. Salzman, R. A., Brady, J. A., Finlayson, S. A., Buchanan, C. D., Summer, E. J., Sun, F., Klein, P. E., Klein, R. R., Pratt, L. H., and Cordonnier-Pratt, M.-M. 2005. Transcriptional profiling of sorghum induced by methyl jasmonate, salicylic acid, and aminocyclopropane carboxylic acid reveals cooperative regulation and novel gene responses. Plant Physiology 138:352-368. 64. Schaller, A., Stintzi, A., and Graff, L. 2012. Subtilases–versatile tools for protein turnover, plant development, and interactions with the environment. Physiologia Plantarum 145:52-66. 65. Scorza, R., Callahan, A., Dardick, C., Ravelonandro, M., Polak, J., Malinowski, T., Zagrai, I., Cambra, M., and Kamenova, I. 2013. Genetic engineering of Plum pox virus resistance: ‘HoneySweet’ plum—from concept to product. Plant Cell, Tissue Organ Culture 115:1-12. 66. Shi, L., Li, R., Liao, S., Bai, L., Lu, Q., and Chen, B. 2014. Prb1, a subtilisin-like protease, is required for virulence and phenotypical traits in the chestnut blight fungus. FEMS Microbiology Letters 359:26-33. 67. Siezen, R. J., de Vos, W. M., Leunissen, J. A., and Dijkstra, B. W. 1991. Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases. Protein Engineering, Design and Selection 4:719-737. 68. Siezen, R. J., and Leunissen, J. A. 1997. Subtilases: the superfamily of subtilisin‐like serine proteases. Protein Science 6:501-523. 69. Singh, A. 2011. Negative feedback through mRNA provides the best control of gene-expression noise. IEEE Transactions on Nanobioscience 10:194-200. 70. Szittya, G., Silhavy, D., Molnár, A., Havelda, Z., Lovas, Á., Lakatos, L., Bánfalvi, Z., and Burgyán, J. 2003. Low temperature inhibits RNA silencing‐mediated defence by the control of siRNA generation. The EMBO Journal 22:633-640. 71. Tinoco, M. L. P., Dias, B. B., Dall'Astta, R. C., Pamphile, J. A., and Aragão, F. J. 2010. In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA. BMC Biology 8:27. 72. Tronsmo, A. 1991. Biological and integrated controls of Botrytis cinerea on apple with Trichoderma harzianum. Biological Control 1:59-62. 73. Ueki, S., and Citovsky, V. 2002. The systemic movement of a tobamovirus is inhibited by a cadmium-ion-induced glycine-rich protein. Nature Cell Biology 4:478. 74. Van Baarlen, P., Staats, M., and Van Kan, J. A. 2004. Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica. Molecular Plant Pathology 5:559-574. 75. Van den Ende, J., Pennock-Vos, M., Bastiaansen, C., Koster, A., and Van der Meer, L. 1998. BoWaS: a weather-based warning system for the control of Botrytis blight in lily. Pages 215-220 in: XXV International Horticultural Congress, Part 9: Computers and Automation, Electronic Information in Horticulture 519. 76. Wang, X., Jiang, N., Liu, J., Liu, W., and Wang, G.-L. 2014. The role of effectors and host immunity in plant–necrotrophic fungal interactions. Virulence 5:722-732. 77. Wolpert, T. J., Dunkle, L. D., and Ciuffetti, L. M. 2002. Host-selective toxins and avirulence determinants: what's in a name? Annual Review of Phytopathology 40:251-285. 78. Wright, C. S., Alden, R. A., and Kraut, J. 1969. Structure of subtilisin BPN’ at 2.5 Å resolution. Nature 221:235. 79. Xia, Y. 2004. Proteases in pathogenesis and plant defence. Cellular Microbiology 6:905-913. 80. Youssef, R. M., Kim, K.-H., Haroon, S. A., and Matthews, B. F. 2013. Post-transcriptional gene silencing of the gene encoding aldolase from soybean cyst nematode by transformed soybean roots. Experimental Parasitology 134:266-274. 81. Zhang, H., Li, H. C., and Miao, X. X. 2013. Feasibility, limitation and possible solutions of RNAi‐based technology for insect pest control. Insect Science 20:15-30. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74550 | - |
dc.description.abstract | LsGRP1 為葵百合的植物防禦相關蛋白,經由水楊酸澆灌處理葵百合,可增加LsGRP1的表現量,並能增加植物對百合灰黴病菌(Botrytis elliptica)的抗性。利用免疫共沉澱法釣取與 LsGRP1 交互作用之百合灰黴病菌蛋白時,找到大小接近 35 kDa 的蛋白,經高效液相層析電灑法串聯式質譜分析,初步鑑定可能為似枯草桿菌蛋白酶(subtilisin-like protease, subtilase),屬於絲胺酸蛋白酶(serine protease),並且在本實驗室建立之感染百合灰黴病菌之百合轉錄體資訊比對到百合灰黴病菌 subtilase 部分序列(劉,2016),因此本研究旨在探討百合灰黴病菌之推定subtilase與 LsGRP1 之間的交互作用關係。首先取得 subtilase 的cDNA序列,命名為 BeSerp。RT-qPCR分析指出BeSerp 基因在葵百合感染灰黴病菌初期有高的表現量但接種後 48小時的表現量下降。利用 BeSerp之dsRNA 處理病菌孢子後接種百合葉,會使百合灰黴病菌感染的病徵較為嚴重。BeSerp之寄主誘導基因靜默(host-induced gene silencing, HIGS)測試也觀察到BeSerp 與 LsGRP1基因表現以及病徵的差異。藉由農桿菌浸潤處理三天,使百合增量表現 BeSerp後接種百合灰黴病菌,葉片之病徵較為輕微。於病毒誘導基因靜默(virus-induced gene silencing, VIGS) LsGRP1 的百合上,BeSerp 表現也有改變。進一步利用pET-29a(+) 表現 BeSerp,將LsGRP1與 BeSerp 蛋白進行生體外共培養,西方墨漬法偵測到不同於原始蛋白大小的條帶,相對分子量分別為40~50 kDa 及10 kDa 左右。基於一系列的研究認為 BeSerp與 LsGRP1 之間存在著某種關聯,進而影響百合灰黴病菌的感染及百合的病徵表現。 | zh_TW |
dc.description.abstract | LsGRP1 is a plant defense-related protein from Lilium ‘Stargazer’. This protein was found previously to be increased by treating with salicylic acid and capable of enhancing Lilium resistance to the infection of Botrytis elliptica. In order to predict what proteins could interact with LsGRP1, co-immunoprecipitation (Co-IP) was performed and the result showed one protein about 35 kDa might interact with LsGRP1N. Analysis of LC-ESI-MS/MS predicted this putative interacting protein to be a subtilisin-like protease (subtilase), and partial amino acid sequence was found in the transcriptome database of B. elliptica-infected Lilium ‘Stargazer’. Thus, this study aimed to examine the interaction between the putative subtilase of B. elliptica and LsGRP1. Firstly, full-length serine protease cDNA was cloned from B. elliptica and named BeSerp. The gene expression of BeSerp after inoculation with B. elliptica at different times on lily was analyzed, and the result showed that BeSerp transcript appeared in a high amount at initial stage but decreased at 48 hr post fungal inoculation. Then, dsRNA interference and host-induced gene silencing were implemented to examine the involvement of BeSerp in B. elliptica-lily interaction. Transient expression of BeSerp in lily by agroinfiltration was performed and the result indicated symptom of the control was more severe than BeSerp treatment. Thus, BeSerp might have elicitor function or generate elicitor. The gene expression of BeSerp was altered on VIGS-LsGRP1 lily post fungal inoculation. These results indicated the gene expression of BeSerp and LsGRP1 were altered by each other. Afterwards, protein expression vector pET-29a(+) was used to express BeSerp which then co-incubated with LsGRP1 in vitro, and the protein signals about 40~50 kDa and 10 kDa different from the original ones were shown. In brief, the relationship between BeSerp and LsGRP1 was indicated from a series of experiments, which might affect the infection process and symptom development caused by B. elliptica. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:42:10Z (GMT). No. of bitstreams: 1 ntu-108-R05633019-1.pdf: 3142944 bytes, checksum: f67dc32900b5c39a4785fd8f74be705d (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 壹、中文摘要 I
貳、英文摘要 II 參、前言 1 肆、前人研究 3 一、百合 3 二、百合灰黴病 3 三、植物富含甘胺酸蛋白質(Glycine-rich proteins, GRPs) 4 四、葵百合之富含甘胺酸防禦相關蛋白 LsGRP1 6 五、絲胺酸蛋白酶 (serine protease) 7 六、枯草桿菌蛋白酶 (subtilases) 8 七、RNA 干擾 (RNA interference, RNAi) 9 八、寄主誘導性基因靜默 (Host-induced gene silencing, HIGS) 10 伍、 材料與方法 12 一、 供試植物之栽培 12 二、 供試真菌之培養、保存與接種源製備 12 1. 供試真菌培養與保存 12 2. 供試真菌之接種源製備 12 三、 擁有目標片段載體的大腸桿菌轉形株篩選與製備 12 1. 大腸桿菌勝任細胞製備 13 2. 大腸桿菌熱休克轉形 13 3. 大腸桿菌轉形株篩選 13 4. 抽取大腸桿菌質體 14 四、 獲取 B. elliptica subtilase 完整序列 14 1. 設計引子 14 2. 真菌接種並抽取 RNA 15 3. 反轉錄與聚合酶連鎖反應 15 4. 目標 DNA 片段膠體回收及純化 15 5. 建構 B. elliptica subtilase 在 pGEM-T easy 定序與預測分析 16 五、 構築目標基因之農桿菌製備與轉形 16 1. 農桿菌勝任細胞製備 16 2. 農桿菌電穿孔法 16 六、 BeSerp 之 dsRNA 處理 B. elliptica 對其感染之影響 17 1. 製備 BeSerp 之 dsRNA 17 2. B. elliptica 前處理 dsRNA 再接種於百合 17 七、 構築寄主誘導基因靜默 (HIGS) 載體與處理百合種球 18 1. B. elliptica BeSerp 部分基因增幅以及純化 18 2. HIGS 載體構築與轉形 18 3. 百合種球浸泡 19 八、 構築短暫表現 BeSerp 轉形株 19 1. B. elliptica BeSerp 基因增幅以及純化 19 2. 構築短暫表現 BeSerp 19 九、 農桿菌浸潤法 20 十、 大腸桿菌 Escherichia coli 表現 BeSerp 融合蛋白 20 1. 大腸桿菌表現蛋白株之建構與保存 20 2. 誘導大腸桿菌表現融合蛋白及蛋白質萃取 21 3. 蛋白質純化及透析 21 4. 冷凍乾燥法濃縮蛋白 22 5. 蛋白質定量 22 十一、 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳偵測蛋白 23 1. 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 23 2. 考馬斯藍染色 23 3. 西方墨漬法 24 十二、 反轉錄-即時聚合酶定量連鎖反應 (Reverse transcription-quantitative PCR, RT-qPCR) 24 十三、 抽取百合葉組織及真菌全基因體核酸 25 十四、 BeSerp在接種之野生型葵百合上的表現量測定 25 十五、 BeSerp 與 LsGRP1 在接種之 HIGS-BeSerp 葵百合上的表現量測定 26 十六、 百合中短暫表現 BeSerp 在有無接種 B. elliptica 後之病徵與基因表現 26 十七、 BeSerp在接種之 VIGS-LsGRP1 葵百合上的表現量測定 26 十八、蛋白酶活性測試 27 十九、 BeSerp 與 LsGRP1ΔSS 蛋白生體外混合試驗 27 陸、結果 28 一、 B. elliptica 絲胺酸蛋白酶功能區預測及與其他物種之枯草桿菌蛋白酶比較 28 二、以有無百合汁液之GB 培養基培養 B. elliptica 後不同時間點之BeSerp表現情形 29 三、 在野生型葵百合上接種 B. elliptica 後不同時間點之 BeSerp表現情形 29 四、前處理BeSerp dsRNA 之 B. elliptica 接種於百合之病徵表現 29 五、 BeSerp 與 LsGRP1 在接種百合灰黴病菌之 HIGS-BeSerp 葵百合上不同時間點之表現量以及病徵比較 30 六、 BeSerp 與 LsGRP1 在百合中短暫表現 BeSerp 之表現狀況 31 七、 BeSerp在接種之 VIGS-LsGRP1 葵百合上不同時間點之表現情形 32 八、 蛋白酶活性測試以及 BeSerp 與 LsGRP1ΔSS 蛋白之體外混合試驗 32 柒、討論 34 捌、參考文獻 41 玖、圖表集 51 表一、用以增幅基因之引子 52 表二、即時定量聚合酶連鎖反應之引子 53 圖一、BeSerp 核酸序列 54 圖二、BeSerp 蛋白預測構造 55 圖三、BeSerp 胺基酸序列與其他菌種之絲胺酸蛋白比對 56 圖四、BeSerp 胺基酸序列與其他菌種之絲胺酸蛋白親源關係樹 57 圖五、以有無百合汁液之GB 培養基培養 B. elliptica後不同時間點之BeSerp表現量 58 圖六、野生型葵百合上接種 B. elliptica 後不同時間點 BeSerp 之表現量 59 圖七、B. elliptica 前處理 dsRNA 再接種百合 60 圖八、BeSerp 在接種之 HIGS-BeSerp 葵百合上不同時間點之表現量測定 61 圖九、LsGRP1 在接種之 HIGS-BeSerp 葵百合上不同時間點之表現量測定 62 圖十、在百合中短暫表現 BeSerp 3天後 BeSerp 與 LsGRP1 表現情形 63 圖十一、農桿菌浸潤後 3 天接種 B. elliptica 之病徵以及 BeSerp 與 LsGRP1 基因表現量比較 64 圖十二、農桿菌浸潤同時接種 B. elliptica 之病徵以及 BeSerp 與 LsGRP1 基因表現量比較 66 圖十三、BeSerp在接種之 VIGS-LsGRP1葵百合上不同時間點之表現量測定 68 圖十四、用於蛋白生產之pET-BeSerp 載體構築 69 圖十五、BeSerp蛋白功能區以及蛋白酶活性測試 70 圖十六、BeSerp 與 LsGRP1 ΔSS 蛋白生體外混合試驗 (I) 71 圖十七、BeSerp 與 LsGRP1 ΔSS 蛋白生體外混合試驗(II) 72 壹拾、附錄 73 圖一、HIGS-BeSerp 載體之構築 74 圖二、農桿菌短暫表現 pBI121-BeSerp 載體之構築 75 | |
dc.language.iso | zh-TW | |
dc.title | 百合灰黴病菌之絲胺酸蛋白酶 BeSerp 與植物防禦相關蛋白 LsGRP1 之關係探討 | zh_TW |
dc.title | Exploration of the relationship between Botrytis elliptica
subtilase BeSerp and plant defense-related protein LsGRP1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 沈偉強,林乃君,黃健瑞,黃祥恩 | |
dc.subject.keyword | 百合灰黴病菌,絲胺酸蛋白?,似枯草桿菌蛋白?,LsGRP1,百合,病毒誘導基因靜默,短暫表現,寄主誘導基因靜默, | zh_TW |
dc.subject.keyword | Botrytis elliptica,serine protease,subtilisin-like protease (subtilase),LsGRP1,lily,virus-induced gene silencing,transient expression,host-induced gene silencing, | en |
dc.relation.page | 75 | |
dc.identifier.doi | 10.6342/NTU201902648 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-07 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
顯示於系所單位: | 植物病理與微生物學系 |
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