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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 曲芳華(Fang-Hua Chu) | |
| dc.contributor.author | Nien-Ting Chiang | en |
| dc.contributor.author | 江念庭 | zh_TW |
| dc.date.accessioned | 2021-06-16T09:55:28Z | - |
| dc.date.available | 2019-02-08 | |
| dc.date.copyright | 2017-02-08 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-12-28 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60091 | - |
| dc.description.abstract | 木酚素 (lignan) 為2個C6-C3單元結合為骨架之植物酚類化合物，具有多樣的生物活性，如臺灣杉 (Taiwania cyptomerioides Hayata) 木材中之dimethylmatairesinol、taiwanin A及taiwanin E 等，即具有顯著毒殺腫瘤細胞之生物活性，可望發展為新的抗癌藥物。Pinoresinol-lariciresinol reductase (PLR) 為木酚素生合成途徑上游酵素，可催化pinoresinol還原成lariciresinol，再將lariciresinol還原成secoisolariciresinol。Secoisolariciresinol經secoisolariciresinol dehydrogenase (SDH) 氧化，生成之產物matairesinol，一般認為是下游木酚素生合成之前驅物。為了解臺灣杉木酚素生合成酵素，本研究建立臺灣杉木材形成層、邊材、移行帶及心材等木材組織轉錄體資料庫，透過分析基因在此4個組織中之表現量相關性，選取3個臺灣杉PLR及6個SDH候選基因，並以cDNA末端快速擴增實驗獲得基因全長。將臺灣杉PLR及SDH基因於大腸桿菌中異源表現，萃取粗蛋白進行酵素反應實驗，其中TcPLR1可催化 (+)-pinoresinol還原為lariciresinol，TcPLR2.2及TcPLR3可催化 (+)-pinoresinol及lariciresinol之還原反應，產生 secoisolariciresinol。RT-PCR結果顯示TcPLR1之表現量於形成層最高，其次為心材，TcPLR2.2及TcPLR3則皆只於形成層及邊材表現，又前者在形成層表現量較高，後者主要於邊材表現。本研究完成臺灣杉PLR之鑑定，未來透過臺灣杉PLR基因之表現趨勢，期望有助於找尋下游木酚素生合成酵素，以建立臺灣杉木酚素之生合成途徑。 | zh_TW |
| dc.description.abstract | Lignans, which are synthesized by dimerization of two C6-C3 units, exist abundantly in the wood of Taiwania cryptomerioides Hayata. Lignans in Taiwania, such as dimethylmatairesinol, taiwanin A and taiwanin E, were reported to have siginificant cytotoxic activity against tumor cells. As the key enzyme of lignan biosynthesis, pinoresinol-lariciresinol reductase (PLR) catalyzes the two-step reduction of pinoresinol to form lariciresinol and then to secoisolariciresinol. Secoisolariciresinol would next be oxidized by secoisolariciresinol dehydrogenase (SDH), and the product matairesinol is suggested to be the precursor for other downstream ignans. In this study, three PLR and six SDH candidate genes of T. cryptomerioides were identified by analysing the transcriptome of wood cambium, sapwood, transition zone and heartwood. The complete coding sequences (CDS) of each gene were obtained by rapid amplification of cDNA ends (RACE). Using Escherichia coli as the host, the heterologously expressed TcPLRs exhibited PLR activity, but the TcSDHs did not exhibited SDH activity. Among the three TcPLRs, TcPLR1 reduced pinoresinol to lariciresinol, while TcPLR2.2 and TcPLR3 reduced both pinoresinol and lariciresinol. The results of RT-PCR revealed that TcPLR1 most highly expressed in the wood cambium, and next in the heartwood. TcPLR2.2 and TcPLR3 both expressed in the wood cambium and the sapwood, while TcPLR2.2 showed slightly higher expression level in the cambium, and TcPLR3 mostly expressed in the sapwood. This study characterizes three PLRs of T. cryptomerioides, which involve in the biosynthesis of upstream lignans, and the expression pattern of TcPLR genes may provide information for identifying downstream lignan biosynthetic genes in the future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T09:55:28Z (GMT). No. of bitstreams: 1 ntu-105-R03625010-1.pdf: 10159142 bytes, checksum: dafd8097e45bd0654f5c87ec79e5aab7 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 口試委員會審定書
誌謝 I 摘要 III Abstract IV 縮寫表 VI 目錄 VII 圖目錄 XIII 表目錄 XVI 第一章 前言 1 第二章 文獻回顧 3 2.1 木酚素 3 2.1.1 木酚素之結構 3 2.1.2 木酚素之功能 3 2.1.3 木酚素之生合成途徑 4 2.1.4 Pinoresinol-lariciresinol reductase之介紹 7 2.1.4.1 受質鏡像選擇性 (enantioselectivity) 7 2.1.4.2 PLR之蛋白質結構 10 2.1.4.3 PLR之蛋白質家族 10 2.1.5 Secoisolariciresinol dehydrogenase之介紹 11 2.1.5.1 受質鏡像選擇性 11 2.1.5.2 SDH之蛋白質結構 12 2.1.5.3 SDH之蛋白質家族 13 2.1.6 木酚素生合成之誘導 14 2.2 臺灣杉 15 2.2.1 臺灣杉簡介 15 2.2.2 臺灣杉木酚素成分及活性研究 15 第三章 材料與方法 18 3.1 實驗試材 18 3.1.1 試材 18 3.1.2 載體之選用 19 3.1.3 菌株之選用 19 3.1.4 藥品與試劑 19 3.2 總量RNA萃取 21 3.3 臺灣杉轉錄體資料庫之建立與基因表現量倍數變化分析 22 3.4 木酚素生合成基因比對 23 3.5 基因表現相關性分析 24 3.6 臺灣杉TcPLRs及TcSDHs基因之選殖 25 3.7 基因與蛋白質序列比對及演化樹分析 27 3.8 蛋白質結構預測 28 3.9 重組蛋白表現 28 3.9.1 表現載體之構築 28 3.9.2 誘導重組蛋白表現與西方墨點試驗 29 3.10 重組蛋白純化 30 3.11 酵素反應 30 3.11.1 粗蛋白反應 30 3.11.2 純化蛋白反應 32 3.12 反應產物分析 32 3.13 木材組織之基因表現量 33 第四章 結果與討論 34 4.1 轉錄體資料庫全新組裝與註解 34 4.2 轉錄體資料庫分析 35 4.2.1 註解為基因之片段重疊群表現於各組織之數目 35 4.2.2 基因表現量倍數變化 39 4.2.3 木酚素生合成酵素所屬之蛋白質超家族 52 4.3 木酚素生合成途徑基因比對與表現量分析 56 4.3.1木質醇 (monolignol) 生合成途徑 56 4.3.2 木酚素生合成途徑 58 4.3.3 基因表現相關性分析 64 4.4 基因序列比對 68 4.4.1 TcPLRs序列比對 68 4.4.2 TcSDHs序列比對 71 4.5 蛋白質序列比對 71 4.5.1 TcPLRs序列比對 71 4.5.2 TcSDHs序列比對 74 4.6 演化樹分析 77 4.6.1 PLR演化樹 77 4.6.2 SDH演化樹 78 4.7 重組蛋白表現 80 4.7.1 重組PLRs表現 80 4.7.2 重組SDHs表現 81 4.8 SDHs重組蛋白純化 82 4.9 酵素反應 83 4.9.1 PLRs酵素反應 83 4.9.2 SDHs酵素反應 83 4.10 蛋白質結構預測 91 4.10.1 TcPLRs結構預測 91 4.10.2 TcSDHs結構預測 93 4.11 木材組織基因表現情形 97 4.11.1 TcPLRs表現情形 97 4.11.2 TcSDHs表現情形 98 第五章 結論 100 引用文獻 101 附錄 111 附錄表1. 表現量於木材形成層及邊材呈現倍數變化之基因於各組織之表現量。 111 附錄表2. 表現量於邊材及移行帶呈現倍數變化之基因於各組織之表現量。 112 附錄表3. 表現量於移行帶及心材呈現倍數變化之基因於各組織之表現量。 114 附錄表4. 註解功能為NAD(P)-binding Rossmann-fold superfamily protein之基因表現量。 116 附錄表5. 註解功能為cytochrome P450之基因表現量。 119 附錄表6. 註解功能為2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein之基因表現量。 122 附錄表7. 用已知的木質醇生合成途徑酵素基因作為模版，比對臺灣杉相應之基因序列。 125 附錄表8. 已知的DP、PLR與SDH基因相對應之臺灣杉基因表現量。 127 附錄表9. 已知的其他木酚素生合成酵素基因相對應之臺灣杉基因表現量。 128 附錄表10. 已知的AtSND1與AtMYB46基因對應之臺灣杉基因表現量。 129 附錄表11. 引子序列表。 130 | |
| dc.language.iso | zh-TW | |
| dc.subject | 木酚素 | zh_TW |
| dc.subject | 臺灣杉 | zh_TW |
| dc.subject | 木材轉錄體 | zh_TW |
| dc.subject | 松脂醇-落葉松脂醇還原? | zh_TW |
| dc.subject | 閉聯異落葉松脂醇脫氫? | zh_TW |
| dc.subject | pinoresinol-lariciresinol reductase | en |
| dc.subject | Taiwania cryptomerioides Hayata | en |
| dc.subject | wood transcriptome | en |
| dc.subject | lignan | en |
| dc.subject | secoisolariciresinol dehydrogenase | en |
| dc.title | 臺灣杉 Pinoresinol-Lariciresinol Reductase之選殖與特性分析 | zh_TW |
| dc.title | Cloning and Characterization of Pinoresinol-Lariciresinol
Reductase from Taiwania cryptomerioides Hayata | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王升陽(Sheng-Yang Wang),何政坤(Cheng-Kuen Ho),孫英玄(Ying-Hsuan Sun),葉汀峰(Ting-Feng Yeh) | |
| dc.subject.keyword | 木酚素,臺灣杉,木材轉錄體,松脂醇-落葉松脂醇還原?,閉聯異落葉松脂醇脫氫?, | zh_TW |
| dc.subject.keyword | lignan,Taiwania cryptomerioides Hayata,wood transcriptome,pinoresinol-lariciresinol reductase,secoisolariciresinol dehydrogenase, | en |
| dc.relation.page | 134 | |
| dc.identifier.doi | 10.6342/NTU201603842 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-12-29 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 森林環境暨資源學研究所 | zh_TW |
| 顯示於系所單位： | 森林環境暨資源學系 | |
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