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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 曲芳華(Fang-Hua Chu) | |
| dc.contributor.author | Kuan-Feng Huang | en |
| dc.contributor.author | 黃冠鳳 | zh_TW |
| dc.date.accessioned | 2021-06-16T13:03:54Z | - |
| dc.date.available | 2015-08-14 | |
| dc.date.copyright | 2013-08-14 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-05 | |
| dc.identifier.citation | 宋家瑋、陸明德、歐錫坤 (2007) 台灣原生山櫻花 (Prunus campanulata Maxim.) 大果種原之選拔。臺灣農業研究56(4): 289-297.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61479 | - |
| dc.description.abstract | 山櫻花 (Prunus campanulata Maxim.) 又名緋寒櫻，為台灣原生的闊葉樹種，分布於全島海拔500-2200公尺之山地闊葉林中，許多公園校園及山區名勝亦有種植。現有關於山櫻花的研究多與其種子休眠和發芽有關，並未見到有關山櫻花之二次代謝物之研究。本研究著眼於山櫻花二次代謝物中之萜類化合物，特別是分子量較小且具有揮發性的單萜和倍半萜，希望從分子生物學的方面探討山櫻花萜類生合成酵素。我們由資料庫中萜類合成酶的基因序列來設計退化性引子對，並且利用次世代定序技術獲得萜類合成酶之片段序列，以反轉錄聚合酶連鎖反應技術，從樹木成熟葉組織中選殖出五組具有典型合成酶保守序列之基因，接著經由5’ 端及3’端快速增幅cDNA端點 (rapid amplification of cDNA ends, RACE) 選殖，獲得完整基因全長。將所得之基因分別構築於蛋白質表現載體中，轉入大腸桿菌 (Escherichia coli) 中表現，發現其中之兩個合成酶可催化單萜及倍半萜前驅物分別生成芳樟醇 (linalool) 和橙花叔醇 (nerolidol)；另外三個萜類合成酶則無活性表現，因此還無法鑑定其生合成之產物。目前已有實驗證實Linalool和Nerolidol皆是與花朵和水果之香味及風味最有關係的萜類之一，除此之外，兩者皆能幫助植物抵抗植食者，並且都具有良好的抗真菌等生物活性。 | zh_TW |
| dc.description.abstract | Prunus campanulata Maxim. also known as Taiwan cherry, is a native broadleaf tree in Taiwan, distributed throughout the island between 500-2200 meters above sea level, and are also planted in many parks, campus and mountain attractions. To date, most of the researches about Prunus campanulata Maxim. are concerned with the seed’s dormancy and germination, but rarely mention about its secondary metabolites. Here we focused on the terpene synthases, especially for those that produce compounds which are small and have volatility. At the beginning, we designed pairs of degenerated primers, and then used it by polymerase chain reaction (PCR) to get the fragments of terpene synthase (TPSs) include the conserve region. Furthermore, we used the next generation sequencing methods to get more fragments that we need, then after 5’ and 3’ Rapid Amplification of cDNA Ends (RACE) done, the full-length cDNA was obtained, with these methods, we obtained five terpene synthase sequences from the leaves of Prunus campanulata Maxim. To identify the function of this five terpene synthases, the recombination protein from Escherichia coli was reacted with gernyl diphosphate (GPP) and farnesyl diphosphate (FPP), they are the substrate of monoterpene and sesquiterpene respectively. Using GC/MS analysis, we revealed that two of these terpene synthases have their major products as linalool and nerolidol. However, the other three terpene synthases do not show any activity. Their functions require further study. Linalool and nerolidol have been confirmed to be the most important terpenes for the fragrance and flavor of flowers and fruits. In addition, these two terpenes both can help plants to own resistance against herbivores, and good bioactivity, including antifungal, anti-inflammatory or anxiolytic etc. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T13:03:54Z (GMT). No. of bitstreams: 1 ntu-102-R99625029-1.pdf: 2771140 bytes, checksum: 69b4aacb0a1bd969f44a243d3431a9ce (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 口試委員審定書………………………………………………………………………i
誌謝……………………………………………………………………………………ii 摘要…………………………………………………………………………………iv Abstract………………………………………………………………………………v 目錄………………………………………………………………………………… vi 圖目錄……………………………………………………………………………… ix 表目錄……………………………………………………………………………… xi 一、前言 …………………………………………………………………………… 1 二、前人研究 ……………………………………………………………………… 3 2.1 萜類化合物之種類 ………………………………………………………… 3 2.2 萜類化合物之生合成路徑 …………………………………………………5 2.3 萜類化合物與植物生理之關聯及其在人類生活之應用 …………………6 2.4 萜類合成酶之基因功能鑑定 ……………………………………………… 9 三、材料方法 …………………………………………………………………… 10 3.1 實驗試材 …………………………………………………………………… 10 3.1.1 植物試材 ………………………………………………………………… 10 3.1.2 載體之選用 ……………………………………………………… 10 3.1.3 菌株之選用 ……………………………………………………… 11 3.2 總量RNA之萃取 …………………………………………………………… 11 3.3 cDNA製備 ………………………………………………………………… 11 3.4 基因選殖 …………………………………………………………………… 11 3.4.1 設計退化性引子對 ………………………………………………………11 3.4.2 基因片段之選殖 ………………………………………………………… 12 3.4.3 次世代定序 …………………………………………………………… 12 3.4.4 基因全長之選殖 ………………………………………………………… 13 3.5 自基因組中選殖含內隱子之基因全長 …………………………………… 13 3.6 序列比對與演化分析 ………………………………………………………14 3.7 蛋白質結構………………………………………………………………… 15 3.8 蛋白質表現 ………………………………………………………………… 15 3.8.1 構築表現載體 …………………………………………………………… 15 3.8.2 誘導表現重組蛋白質 …………………………………………………… 16 3.8.3 蛋白質電泳 ……………………………………………………………… 16 3.8.4 免疫雜合反應 ……………………………………………………………16 3.9 活性反應 …………………………………………………………………… 17 3.9.1 蛋白質之生成及反應 …………………………………………………… 17 3.9.1.1粗蛋白 (crude protein) 反應 ……………………………………………… 17 3.9.1.2均質蛋白質 (homogeneous) 反應 ……………………………………… 17 3.9.2 氣象層析質譜分析儀 (GC-MS) 進行產物鑑定 ………………………… 18 3.10 各組織mRNA表現量…………………………………………………………… 18 3.11 傷害處理………………………………………………………………………… 19 3.11.1 化學性傷害處理 …………………………………………………… 19 3.11.2 物理性傷害處理 …………………………………………………… 19 3.11.3 反轉錄聚合酶連鎖反應 …………………………………………… 20 四、實驗結果 ………………………………………………………………… 21 4.1 基因全長之選殖 ………………………………………………………… 21 4.2 序列比對 ………………………………………………………………… 22 4.3 基因組結構分析 ………………………………………………………… 25 4.4 相似性樹狀圖分析 ……………………………………………………… 27 4.5 蛋白質結構預測 ………………………………………………………… 29 4.6 表現重組蛋白質 ………………………………………………………… 33 4.7 重組蛋白質活性反應 ……………………………………………………… 36 4.8 山櫻花萜類合成酶於各組織之基因表現 ………………………… 39 4.9 經過傷害處理之山櫻花萜類合成酶基因表現 ………………………… 41 五、討論………………………………………………………………………… 42 5.1 山櫻花萜類合成酶序列特性 …………………………………………… 42 5.2 未表現功能之萜類合成酶 ……………………………………………… 44 5.3 linalool與nerolidol之生合成路徑 ……………………………………… 46 5.4 萜類產物之生物活性 …………………………………………………… 47 5.5 基因組結構分析 ………………………………………………………… 48 5.6 相似性樹狀圖分析 ……………………………………………………… 50 5.7 萜類合成酶於不同組織之表現 ………………………………………… 51 5.8 傷害處理對基因表現量的影響 ………………………………………… 52 六、結論 …………………………………………………………………… 53 七、引用文獻 ………………………………………………………………… 54 八、附錄 ……………………………………………………………………… 67 附錄一、引子序列表 …………………………………………………… 67 附錄二、圖七物種學名對照表 ………………………………………… 71 | |
| dc.language.iso | zh-TW | |
| dc.subject | 芳樟醇 | zh_TW |
| dc.subject | 倍半?合成? | zh_TW |
| dc.subject | 山櫻花 | zh_TW |
| dc.subject | 單?合成? | zh_TW |
| dc.subject | 橙花叔醇 | zh_TW |
| dc.subject | sesquiterpene synthase | en |
| dc.subject | linalool | en |
| dc.subject | nerolidol | en |
| dc.subject | monoterpene synthase | en |
| dc.subject | Prunus campanulata Maxim. | en |
| dc.title | 山櫻花萜類合成酶基因之選殖與表現 | zh_TW |
| dc.title | Cloning and Expression of Terpene Synthase Genes
From Taiwan Cherry (Prunus campanulata) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王升陽(Sheng-Yang Wang),楊健志(Chien-Chih Yang),張淑華(Shu-Hwa Chang),鍾國芳(Kuo-Fang Chung) | |
| dc.subject.keyword | 山櫻花,芳樟醇,橙花叔醇,單?合成?,倍半?合成?, | zh_TW |
| dc.subject.keyword | Prunus campanulata Maxim.,monoterpene synthase,sesquiterpene synthase,linalool,nerolidol, | en |
| dc.relation.page | 71 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-08-05 | |
| 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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