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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭貽生 | |
| dc.contributor.author | Yung-Chi Huang | en |
| dc.contributor.author | 黃詠琪 | zh_TW |
| dc.date.accessioned | 2021-06-16T10:18:05Z | - |
| dc.date.available | 2018-08-28 | |
| dc.date.copyright | 2013-08-28 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-17 | |
| dc.identifier.citation | 邱宜芳 (2003) 甘藷傷害誘導之蛋白質Ipomoelin的功能測定。國立台灣大學植物學研究所碩士論文。
張維倢 (2011) 甘藷凝集素ipomoelin四級結構與多種醣類之熱力學分析顯示其多變的結合特性。國立臺灣大學植物科學研究所碩士論文。 Abecasis, A.B., Geretti, A.M., Albert, J., Power, L., Weait, M., and Vandamme, A.M. (2011). Science in court: the myth of HIV fingerprinting. Lancet. Infect. Dis. 11: 78-79. Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Echols, N., Headd, J.J., Hung, L.W., Jain, S., Kapral, G.J., Kunstleve, R.W.G., McCoy, A.J., Moriarty, N.W., Oeffner, R.D., Read, R.J., Richardson, D.C., Richardson, J.S., Terwilliger, T.C., and Zwart, P.H. (2011). The Phenix software for automated determination of macromolecular structures. Methods. 55: 94-106. Allsopp, P.G., and McGhie, T.K. (1996). Snowdrop and wheatgerm lectins and avidin as antimetabolites for the control of sugarcane whitegrubs. Entomol. Exp. Appl. 80: 409-414. Bailey, T.A., Samour, J.H., Sheen, R.S., and Garner, A. (1996). Analysis of amoxycillin in bustard plasma by high-performance liquid chromatography after administration of a long-acting formulation. J. Vet. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60434 | - |
| dc.description.abstract | 甘藷凝集素Ipomoelin(IPO)為甘藷受到傷害時，在葉子誘導表現的植物性防禦蛋白質。根據文獻結果，植物凝集素(lectins)可用來抵抗病毒、細菌、真菌以及昆蟲。IPO是屬於mannose-specific jacalin-related lectin(mJRL)次家族的醣結合蛋白，但亦能與半乳糖(galactose)結合，顯示其具有多種醣類結合的能力。IPO晶體結構呈現四聚體形式，每個單體N端具兩個短鏈的β摺板，約10個胺基酸，具有改變對醣類的結合能力，並且和IPO形成不同的多聚體相關聯。本研究進一步對於IPO的N端序列及其抗蟲抗病能力進行探討，首先構築了N端去掉前6個胺基酸的IPO(ND6)，相當去掉N端一個β摺板，晶體結構及膠體過濾色層分析法(gel filtration chromatography)結果顯示ND6為二聚體。進一步比較wtIPO(四聚體)和ND10(單體)，利用等溫滴定微熱量法(isothermal titration calorimetry, ITC)得知ND6對甲基化甘露醣(methyl-α-D-mannopyranoside, Me-Man)的結合能力介於wtIPO和ND10之間，呈現過渡態的形式。IPO和不同物種同屬JRL家族的凝集素序列比對的結果，將醣結合區域(carbohydrate binding pocket )之Trp-142位置定位點突變(site-directed mutagenesis)成其他物種之胺基酸，利用ITC測試其醣結合能力是否改變，以確定Trp-142是穩定甲基化單醣結合的重要胺基酸。在進行家蠶餵食實驗結果顯示，抑蟲能力為ND10 > ND6 > wtIPO；而在抑制大腸桿菌(Inhibition assay)的實驗中，抑菌能力為wtIPO > ND6 > ND10。利用差異性掃描螢光法(Differential scanning fluorimetry, DSF)測定變性溫度(melting temperature, Tm) 得知IPO不同蛋白質之熱穩定性，而wtIPO 在不同的酸鹼值之下會比ND6 及ND10來得相對穩定；利用次細胞定位(subcellular localization)得知IPO不只會存在在細胞質中，也會出現於細胞核中，代表其有可能具有訊息傳遞的功能。 | zh_TW |
| dc.description.abstract | Ipomoelin (IPO), a plant defense protein, is induced by mechanical wounding or herbivorous attacking. It was isolated from sweet potato (Ipomoea batatas cv. Tainung 57) and belongs to mannose-specific jacalin-related lectin (mJRL) family. The quaternary structure of IPO reveals a tetrameric association. However, previous results indicated that IPO could also bind to galactose and showed more diverse carbohydrate binding ability than that of mJRL. The first 10 amino acids of N-terminus of IPO are important to carbohydrate binding and might be involved in different oligomerization of IPO. Several experiments were used to identify the roles of N-terminus of IPO. A mutant protein with truncated N-terminal 6 residues of IPO (ND6) was constructed. The dimeric form of ND6 was identified by X-ray crystallography and size exclusion chromatography (SEC). In comparison with wtIPO (tetramer form) and ND10 (monomer form), the binding affinity of ND6 to methyl-α-D-mannopyranoside (Me-Man) showed its transition manner in the middle of wtIPO and ND10 by isothermal titration calorimetry. According to the sequence alignment between IPO and other JRL family lectins, we use site-directed mutagenesis change the Trp-142 of IPO to other lectins residue. Use the ITC studies to confirm the residue Trp-142 as a key residue for stabilizing the binding of methylated carbohydrates. In the silkworm feeding experiment, the retarded growth ability is ND10 > ND6 > wtIPO. For the antibacterial assay, the inhibition growth of Escherichia coli is wtIPO > ND6 > ND10. The melting temperature determined by differential scanning fluorimetry (DSF) showed that wtIPO was more stable in wide range of pH than those of ND6 and ND10. Furthermore, wtIPO would exist in both cytoplasm and nucleus by subcellular localization experiment. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T10:18:05Z (GMT). No. of bitstreams: 1 ntu-102-R00b42016-1.pdf: 8453607 bytes, checksum: d3846139e7cf1d3b1a94c63ccb8d872f (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 目錄 I
中文摘要 V Abstract VI 縮寫對照表 VII 第一章 前言 1 一、 甘藷凝集素Ipomoelin簡介 1 二、 植物凝集素 2 三、 波羅蜜相關凝集素 3 四、 植物凝集素之抗蟲及抗菌能力 5 五、Ipomoelin(IPO)蛋白質功能分析 7 六、Ipomoelin(IPO)之蛋白質結構 8 七、Ipomoelin(IPO)之醣結合區域 8 八、植物凝集素於細胞中的位置 9 九、研究目標 9 第二章 材料與方法 11 一、材料 11 二、方法 11 （一）IPO基因表現載體之構築 11 1. ND6、ND10表現載體之構築 11 2. IPO定位點突變基因表現載體之構築 11 （二）IPO蛋白質之表現與純化 12 1. 大腸桿菌勝任細胞之製備 12 2. 大腸桿菌之轉型作用 12 3. 重組蛋白質之大量表現與純化 12 4. SDS-聚丙烯醯胺膠體電泳 13 5. 蛋白質之定量分析 14 （三）IPO ND6蛋白質結晶實驗與結構分析 14 1. Pre-crystallization test 14 2. IPO ND6蛋白質之結晶篩選 14 3. X-ray 繞射晶體數據之收集與整理 15 4. IPO ND6蛋白質結構之決定與精調 15 5. IPO ND6蛋白質結構驗證與繪製 16 （四）wtIPO、ND6、ND10蛋白質之多聚體組成分析 16 （五）IPO及其突變蛋白質與醣類結合能力之分析 17 1. 蛋白質樣品的製備和定量 17 2. 醣類溶液的配製 17 3. 等溫滴定微熱量法(ITC) 18 （六）測定IPO蛋白質熱穩定性及不同酸鹼度之穩定性 19 （七）家蠶餵養實驗 19 （八）抑制病原菌之能力分析 20 1. 大腸桿菌之抑制 20 2. 番茄細菌性斑點菌之抑制 20 3. 細菌軟腐病菌之抑制 21 （九）次細胞定位 21 1. IPO螢光表現載體之構築 21 2. 萃取質體DNA 22 3. 分離阿拉伯芥原生質體 22 4. 原生質體的基因轉殖 23 5. 利用共軛焦顯微鏡觀察螢光蛋白質表現之區域 23 第三章 結果 24 一、IPO 蛋白質及不同突變蛋白質之表現與純化 24 二、IPO ND6晶體及結構分析 25 三、wtIPO及ND6、ND10蛋白質之多聚體組成分析 26 四、IPO及突變蛋白質與醣類結合能力之分析 27 五、IPO之熱穩定性及不同酸鹼度之下的穩定性 28 六、抗蟲能力測試 29 七、抑制病原菌之能力分析 30 八、次細胞定位 31 第四章 討論 32 一、IPO蛋白質之表現與純化 32 二、ND6晶體結構分析 32 三、wtIPO、ND6、ND10多聚體組成探討 33 四、wtIPO、ND6、ND10及不同突變蛋白質醣結合能力探討 33 五、wtIPO、ND6、ND10熱穩定性及酸鹼值穩定性之探討 35 六、wtIPO、ND6、ND10抑蟲及抑菌能力之探討 36 七、IPO細胞內位置之探討 38 第五章 結論 39 參考文獻 41 圖表 51 表一、IPO ND6蛋白質晶體之繞射數收據收集統計資料 51 表二、wtIPO、IPO ND6、IPO ND10、與各種甲基化單醣之熱力學參數* 52 表三、各種定位點突變蛋白質與各種甲基化單醣之結合常數(KA) 52 表四、IPO及其他JRL家族成員與各種單醣交互作用之KA值對照表 53 表五、Ipomoelin不同蛋白質在中性緩衝溶液之變性溫度(Tm) 53 圖一、JRL家族成員之多聚體組成 54 圖二、IPO蛋白質結構 55 圖三、IPO蛋白質結構與其他JRL家族同屬於四聚體結構比較 56 圖四、IPO與Me-Man醣類複合體結構 57 圖五、His-IPO蛋白質之表現與純化 58 圖六、His-ND6蛋白質之表現與純化 59 圖七、His-ND10蛋白質之表現與純化 60 圖八、不同定位點突變蛋白質之表現純化 61 圖九、IPO ND6蛋白質之晶體 62 圖十、IPO ND6之結構 解析 63 圖十一、分子量標準品膠體過濾色層分析 64 圖十二、wtIPO、 ND6、ND10之多聚體分析 65 圖十三、ND6與各種甲基化單醣之ITC分析 66 圖十四、W142D與各種甲基化單醣之ITC分析 67 圖十五、W142K與各種甲基化單醣之ITC分析 68 圖十六、W142Y與各種甲基化單醣之ITC分析 69 圖十七、142Y143W與各種甲基化單醣之ITC分析 70 圖十八、IPO蛋白質其熱穩定性及不同酸鹼值下之穩定性 71 圖十九、家蠶餵養實驗1 (wtIPO、ND10) 72 圖二十、家蠶餵養實驗2 (wtIPO、ND6、ND10) 73 圖二十一、wtIPO、ND6、ND10抑菌能力測試 74 圖二十二、Ipomoelin於阿拉伯芥原生質體中的位置 75 附錄 76 附錄一、表現載體之構築所使用之引子 76 附錄二、聚合酶連鎖反應機器設定 77 附錄三、wtIPO、ND6、ND10蛋白質以及突變蛋白質純化之試劑 78 附錄四、SDS-PAGE膠體電泳之試劑 79 附錄五、Ipomoelin分子量及消光係數 80 附錄六、蛋白質結晶濃度測試之PCT試劑 80 附錄七、中性緩衝溶液Gel filtration buffer 80 附錄八、ITC Buffer 81 附錄九、DSF測定蛋白質在不同酸鹼值之下穩定性之溶液 81 附錄十、原生質體製備之試劑 82 附表一、植物凝集素之分類及在細胞中分布的位置 84 附表二、植物凝集素之分類及抗蟲能力 85 附圖一、甘藷IPO基因受誘導表現之訊息調控路徑 86 附圖二、pTZ18U載體圖譜 87 附圖三、pET28a表現載體圖譜 88 附圖四、IPO與已知結構之植物凝集素蛋白質一級結構序列比對 89 附圖五、差異性螢光掃描法(DSF) 90 | |
| dc.language.iso | zh-TW | |
| dc.subject | 多聚體 | zh_TW |
| dc.subject | 凝集素 | zh_TW |
| dc.subject | 甘藷凝集素 | zh_TW |
| dc.subject | 膠體過濾色層分析 | zh_TW |
| dc.subject | 等溫滴定微量熱法 | zh_TW |
| dc.subject | isothermal titration calorimetry | en |
| dc.subject | jacalin | en |
| dc.subject | oligomerization | en |
| dc.subject | ipomoelin | en |
| dc.subject | gel filtration chromatography | en |
| dc.subject | lectin | en |
| dc.title | 不同聚合形式之甘藷凝集素其抗菌和抗蟲機制之研究 | zh_TW |
| dc.title | Molecular function of Ipomoelin in different oligomerizations for antibacterial and insecticidal ability | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐駿森,張世宗,鄭石通,蔡麗珠 | |
| dc.subject.keyword | 甘藷凝集素,凝集素,多聚體,膠體過濾色層分析,等溫滴定微量熱法, | zh_TW |
| dc.subject.keyword | lectin,jacalin,oligomerization,ipomoelin,gel filtration chromatography,isothermal titration calorimetry, | en |
| dc.relation.page | 90 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-08-17 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| 顯示於系所單位： | 植物科學研究所 | |
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