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dc.contributor.advisor | 黃鵬林(Pung-Ling Huang) | |
dc.contributor.author | Chih-Yao Hsu | en |
dc.contributor.author | 許智堯 | zh_TW |
dc.date.accessioned | 2021-06-08T06:24:38Z | - |
dc.date.copyright | 2006-07-31 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-27 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25683 | - |
dc.description.abstract | 摘要
本論文選殖香椿之萜類合成酶基因,並分析其酵素功能。萜類 (terpenoids) 化合物是植物相當重要的二次代謝產物,雖然它與植物的生長發育並沒有直接的關係,但對植物與環境之間交互作用 (例如:抗寒、缺水等逆境),及植物本身防禦病蟲害是很重要的。萜類合成酶 (terpene synthase, TPS) 是萜類生合成反應過程中最關鍵的酵素。本論文描述從香椿 (Toona sinene) 葉片之中選殖出命名為TsTPS1的TPS基因,其全長為1788個鹼基對,會轉譯出595個胺基酸,預測分子量為68.82 kDa;另構築一條基因,將TsTPS1基因前端運輸蛋白 (transit peptide) 之序列切除,命名為TsTPS1-t,其全長為1617鹼基對,會轉譯出538個胺基酸,預測分子量為62.99 kDa。利用大腸桿菌分別表現前述兩條基因,將所得到的蛋白質經過酵素純化及氣相層析質譜儀 (GC-MS) 定性後,證明從香椿中所選殖出的TsTPS1-t基因會轉譯成單萜類的正檸檬精油合成酶 ( (+)-limonene synthase);TsTPS1基因表現出的酵素量低,經活性測試亦尚未測得酵素活性。以反轉錄聚合酶連鎖反應 (RT-PCR) 的試驗結果顯示TsTPS1基因在香椿根部的表現量較高。TsTPS1基因組DNA (genomic DNA) 全長2317個鹼基對,其中包含了六個顯子 (exon) 與五個隱子 (intron)。從組織的石蠟切片,觀察到香椿葉柄上有高密度的腺細胞 (約10 granules/mm2),在葉身上則未發現任何腺細胞的存在;不過我們觀察到葉片的海綿組織靠近氣孔周圍有儲存油質的細胞。分析香椿葉身正己烷萃取物顯示,香椿的幼葉片中共含31種化合物,其中2種為單萜類、22種為倍半萜類、1種為雙萜類及6種非萜類成分。 | zh_TW |
dc.description.abstract | We cloned and functionally characterized a terpene synthase gene from Chinese Mahogany (Toona sinensis). Terpenoids are one of the important secondary metabolites in plants. Although terpenoids are not directly related to the growth and development of plants, they are important for plants to defense pathogens and to interact with their environments. Terpene synthase (TPS) is the key enzyme in the biosynthesis of terpenoid compounds. A monoterpene gene, TsTPS1, was cloned from T. sinensis. The gene is 1788 bp long and encodes a polypeptide of 595 amino acids which has a predicted molecular weight of 68.82 kDa. A truncated gene, TsTPS1-t, was obtained from TsTPS1. It is 1617 bp long and encodes a polypeptide of 538 amino acids which has a predicted molecular weight of 62.99 kDa. E. coli was used to express TsTPS1 and TsTPS1-t genes. Purified recombinant enzymes were characterized by Gas Chromatograph / Mass Spectrometer (GC/MS). Our assay demonstrated that the TsTPS1-t could be translated into (+)-limonene synthase. RT-PCR test indicated that TsTPS1 is expressed more abundantly in root than other tissues. The gemomic DNA sequence of TsTPS1 is 2317 bp long including six exons and five introns. In paraffin section, we found high density of glandular cells (10 granules/ mm2) in the petiole but not in the leaf blades of T. sinensis. Many oil containing cells near the stoma were found in the leaves of T. sinensis. By extracting with hexane, the total extractants of Chinese Mahogany’s leaves contain 31 kinds of compounds, which are consisted of 2 types of monoterpenoids, 22 types of sesquiterpenoids, 1 type of diterpenoids, and 6 types of non-terpenoids. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T06:24:38Z (GMT). No. of bitstreams: 1 ntu-95-R93628138-1.pdf: 1795627 bytes, checksum: 68dbb605be33d056af27ed527c9b4c2a (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 中文摘要 5
英文摘要 6 壹、 前言 7 貳、 前人研究 9 ㄧ、萜類的生合成 9 二、萜類合成酶胺基酸序列上的特性 10 三、應用 11 (ㄧ)、轉殖linalool synthase基因至康乃馨 11 (二)、轉殖monoterpene synthases基因至菸草中 12 (三)、E-DMNT引起植物的自我防衛機制 13 (四)、玉米利用sesquiterpenoids來吸引草食動物的自然天敵 13 (五)、紫杉醇 (Taxol) 在醫學上的應用 14 (六)、其它 15 參、 材料與方法 16 ㄧ、材料 16 二、試驗方法 16 (一)、RNA之抽取 16 (二)、基因組DNA (genomic DNA) 之抽取 17 (三)、製備double-strand cDNA (ds-cDNA) 17 (四)、退化引子 (degenerate primers) 的設計 18 (五)、基因部分片段增幅及選殖 18 (六)、核酸序列定序及比對 21 (七)、設計專一引子 (specific primers) 21 (八)、三端與五端快速增幅互補DNA (Rapid Amplification of cDNA Ends, RACE) 21 (九)、pET21b表現載體的構築 22 (十)、TsTPS1基因表現及純化 24 (十一)、TsTPS1t基因表現及純化 24 (十二)、聚丙烯醯胺膠體電泳 (SDS-PAGE analysis) 25 (十三)、西方墨點法 (Western blot analysis) 26 (十四)、蛋白質定量 26 (十五)、TsTPS1酵素活性測試及產物分析 27 (十六)、TsTPS1-t酵素活性測試及產物分析 27 (十七)、反轉錄聚合酶鏈連鎖反應 (RT-PCR) 28 (十八)、親緣關係樹 (phylogenetic tree) 之重建 29 (十九)、基因組DNA (genomic DNA) 的定序 29 (二十)、香椿組織切片 29 (二十一)、香椿葉片成分分析 31 肆、 結果 33 一、TSTPS1基因部分片段增幅 33 二、TSTPS1基因的RACE PCR 33 三、表現載體PET21b-TSTPS1的構築 33 四、表現載體PET21b-TSTPS1-t的構築 34 五、質體PET21b-TSTPS1的大量表現及酵素純化 34 六、質體PET21b-TSTPS1-t的大量表現及酵素純化 34 七、TSTPS1酵素活性測試 35 八、TSTPS1-t蛋白質定量 35 九、TSTPS1-t酵素活性測試 36 十、反轉錄聚合酶連鎖反應 (RT-PCR) 36 十一、親緣關係樹之重建 37 十二、基因組DNA (genomic DNA) 定序 37 十三、香椿成熟葉片及葉柄組織切片 37 十四、香椿葉片成分分析 37 伍、 討論 39 ㄧ、(+)-limonene之特性與功能 39 二、香椿之(+)-limonene synthase基因結構特性 39 三、香椿之(+)-limonene synthase酵素活性 40 四、(+)-limonene synthase基因於香椿各部位表現情形 41 五、香椿葉片二次帶謝產物分析 41 六、香椿成熟葉片及葉柄石蠟切片 42 陸、 結論 43 柒、 参考文獻 44 圖表目次 圖一、萜類生合成路徑…………………………………………………………48 圖二、芸香科植物單萜類合成酶基因之胺基酸序列比較……………………………49 圖三、TsTPS1基因全長選殖……………………………………………………50 圖四、中間質體pT-TsTPS1之構築……………………………………………51 圖五、香椿單萜類合成酶基因 (TsTPS1)核苷酸及演譯胺基酸序列…………………52 圖六、蛋白表現質體pET21b-TsTPS1 之構築…………………………………53 圖七、蛋白表現質體pET21b-TsTPS1-t 之構築………………………………54 圖八、利用SDS-PAGE 與西方墨點法偵測香椿TsTPS1重組蛋白的表現與純化…………………………………………………………………………………55 圖九、利用SDS-PAGE 與西方墨點法偵測香椿TsTPS1-t重組蛋白的表現與純化…………………………………………………………………………………56 圖十、利用氣相層析儀與質譜儀 (GC/MS)分析TsTPS1-t的產物……………57 圖十一、檸檬精油鏡相異構物之氣相層析儀分析……………………………58 圖十二、香椿TsTPS1基因於植株不同部位之表現……………………………59 圖十三、重建香椿TsTPS1與32條裸子植物單萜類合成酶胺基酸序列之親緣關係樹……………………………………………………………………………60 圖十四、香椿TsTPS1之基因組DNA序列……………………………………62 圖十五、香椿成熟葉片石蠟切片………………………………………………63 圖十六、香椿葉柄石蠟切片……………………………………………………64 圖十七、香椿葉片總萃取物成分分析…………………………………………65 表一、TsTPS1利用NCBI之BlastX比對結果…………………………………………66 表二、香椿葉片成分分析結果…………………………………………………67 | |
dc.language.iso | zh-TW | |
dc.title | 香椿單萜類合成酶基因之選殖及其功能分析 | zh_TW |
dc.title | Cloning and Functional Analysis of A Monoterpene Synthase Gene in Toona sinensis | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 杜宜殷(Yi-Yin Do),趙淑妙(Shu-Miaw Chaw) | |
dc.contributor.oralexamcommittee | 許圳塗(Chouu-Ton Shii) | |
dc.subject.keyword | 香椿,單萜,類,萜,類合成酶, | zh_TW |
dc.subject.keyword | Toona sinensis,terpenoid,terpene synthase, | en |
dc.relation.page | 70 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2006-07-29 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 園藝學研究所 | zh_TW |
Appears in Collections: | 園藝暨景觀學系 |
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