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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 葉汀峰(Ting-Feng Yeh) | |
| dc.contributor.author | Hsin-Tzu Wang | en |
| dc.contributor.author | 王心慈 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:36:04Z | - |
| dc.date.available | 2017-08-19 | |
| dc.date.copyright | 2012-08-19 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-14 | |
| dc.identifier.citation | Albersheim, P., A. Darvil, K. Roberts, R. Sederoff and A. Staehelin. 2011. Plant Cell Walls. Garland Science, Taylor & Francis Group, New York, 430 p.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64233 | - |
| dc.description.abstract | 由於石油資源日漸枯竭，因此開發再生的替代能源一直是重要的研究課題。生質乙醇(Bioethanol)為目前重要的再生能源之一，可藉由發酵木質纖維中的單糖來製成。唯目前工業發酵的製程尚以利用六碳糖(Hexose)為最有效率的方法，因此利用基因調控來提高木質纖維中六碳糖的含量將有助於能源作物的開發與利用。甘露聚醣(Mannan)為細胞壁多醣之一，是裸子植物細胞壁中含量最多的非纖維素多醣(含量約20%)，同時也是被子植物細胞壁非纖維素多醣的次要組成分之一(含量2 - 5%)，其單糖的六碳結構非常適合作為生質乙醇生產的材料。本研究首先以Mannan之專一性抗體LM21對野生型灰楊(Populus trichocarpa)不同節間之莖部組織進行免疫標定，以觀察Mannan於植物生長過程中的累積情形。結果顯示Mannan在植物發育初期，最先堆積於初生木質部（Primary xylem），並隨著植株的次級生長而逐漸累積於木質部及韌皮纖維（Phloem fiber）的次級細胞壁中。另一方面，LM21的訊號也會受到果膠質(Pectin)及乙醯基(Acetyl group)的遮蔽，但不受木質化(Lignification)及木聚醣(Xylan)堆積的影響。本研究另以轉基因方式利用35S啟動子(Promoter)正向調控灰楊Mannan生合成基因PtiCSLA1(Cellulose synthase-like A1)表現，並以即時定量聚合酶鏈鎖反應(Quantitative real time polymerase chain reaction, qRT-PCR)分析基因表現量，以酵素連結免疫吸附試驗(Enzyme-linked immunosorbent assay, ELISA)分析酵素活性，再以醣類分析及免疫標定技術觀察野生型及轉基因型植株細胞壁多醣的組成及分布。醣類分析的結果顯示，利用單一35S啟動子正向調控PtiCSLA1基因表現的植株，其Mannan含量最高可達到野生型植株的1.5倍。同時免疫標定的結果顯示轉殖株的Mannan含量確實增加，證實灰楊PtiCSLA1基因參與Mannan的生合成。 | zh_TW |
| dc.description.abstract | Due to fossil fuel reserve will be completely depleted, it has been an important issue to seek any alternative and renewable source for fuels. Bioethanol is regarded as the most potential alternative for fossil fuel substitutes. Bioethanol can obtain from fermentation of sugars in lignocelluloses. For current industrial-based fermentation, hexoses are the preferred substrates for ethanol production. Mannan polysaccharides are the most abundant non-cellulosic cell wall polysaccharides in gymnosperm (~20%), however they are minor non-cellulosic polysaccharides in angiosperm (2 - 5%). Mannose is one of hexose members and suitable for bioethanol production. In this study, mannan specific antibody LM21 was used to investigate mannan deposition in wild-type black cottonwood (Populus trichocarpa) stems from different internodes. Results of immunolabeling reveal that mannan deposits in primary xylem in early developmental stages, and then accumulates in secondary walls of xylem cells and phloem fibers during wood formation. On the other hand, LM21 signals can be masked by pectin and acetyl group, but are less affected by lignification and xylan deposition. To investigate the function of PtiCSLA1 (Cellulose synthase-like A1), 35S promoter was used to overexpress PtiCSLA1 in Populus trichocarpa. Gene expression were analyzed by qRT-PCR (Quantitative real time polymerase chain reaction), and enzyme activities were analyzed by enzyme-linked immunosorbent assay (ELISA), and wood compositions were analyzed through carbohydrate analysis and immunolabeling. Results of carbohydrate analysis revealed that mannan contents of PtiCSLA1 transgenic lines driven by single 35S promoter were higher than wild-type plants, and the highest content to be 1.5 fold of that in wild-type plants. Results of immunolabeling by mannan specific antibody also revealed that LM21 signals in transgenic lines were stronger than that of wild-type plants, which provides a strong evidence to support PtiCSLA1 gene involving in mannan biosynthesis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:36:04Z (GMT). No. of bitstreams: 1 ntu-101-R98625008-1.pdf: 4863359 bytes, checksum: ce888656c2ef188fd10331e21bfeffad (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 誌謝 I
摘要 II Abstract III 目錄 V 圖目錄 VIII 表目錄 XV 縮寫說明 XVII 第一章、 前言 1 第二章、 文獻回顧 3 2.1 木質纖維多醣之生合成 3 2.2 CSLA基因表現 7 2.3 甘露聚醣在植物體中的功能及可能扮演的角色 10 2.4 木材性質改質之目的 12 2.5 以免疫標定技術分析細胞壁多醣之組成 14 2.5.1 單株抗體 14 2.5.2 醣類結合模組 20 第三章、 材料與方法 23 3.1 植物材料及生長條件 23 3.2 載體（Vector） 23 3.3 菌株 24 3.4 單株抗體及醣類結合模組 24 3.5 水解酵素 24 3.6 PtiCSLA1基因轉殖株之建立 25 3.6.1 灰楊PtiCSLA1基因專一性序列片段之選殖 25 3.6.2 表現質體之建構 26 3.6.3 灰楊及菸草組織之轉殖 26 3.7 灰楊轉基因植株之確立 28 3.8 PtiCSLA1基因表現量分析 28 3.8.1 灰楊木質部組織及菸草葉部組織總量 RNA的萃取 28 3.8.2 即時定量聚合酶鏈鎖反應（Quantitative real time PCR, qRT-PCR） 29 3.8.3 反轉錄聚合酶鏈鎖反應（Reverse transcription PCR, RT-PCR） 29 3.9 PtiCSLA1蛋白質表現 30 3.9.1 木質部PtiCSLA1之萃取與純化 30 3.9.2 PtiCSLA1專一性抗體的製備 31 3.9.3 西方墨點法（Western blot） 31 3.9.4 PtiCSLA1酵素活性分析 32 3.10 免疫螢光標定分析細胞壁醣類分布 32 3.10.1 莖部組織的包埋與切片 32 3.10.2 切片之前處理 33 3.10.3 免疫螢光標定 33 3.11 莖部組織切片之組織化學染色 34 3.11.1 Phloroglucinol-Hydrochloric acid staining 34 3.11.2 Safranin-O and Fast Green double staining 34 3.12 木材組成分分析 35 3.12.1 木粉之醇苯萃取 35 3.12.2 木質素含量分析 35 3.12.3 醣類含量分析 36 3.13 統計分析 36 第四章、 結果與討論 37 4.1 野生型灰楊樹莖部組織之細胞壁醣類分布 37 4.1.1 不同生長時期之莖部組織分析 37 4.1.2 前處理對莖部組織免疫螢光標定之影響 40 4.2 灰楊轉殖株木質部組織之PtiCSLA1基因表現量分析 48 4.2.1 PtiCSLA1轉基因灰楊的確立 48 4.2.2 PtiCSLA1基因表現量 49 4.3 PtiCSLA1抗血清之專一性測試及酵素活性分析 51 4.4 灰楊轉殖株之木質部細胞壁組成分分析 53 4.4.1 細胞壁醣類組成分析 53 4.4.2 木質素含量分析 56 4.4.3 灰楊轉殖株莖部切片之組織化學染色及免疫螢光標定 57 4.5 野生型灰楊及PtiCSLA1轉殖株其木質部細胞壁組成分之相關分析 64 4.6 菸草轉殖株之PtiCSLA1基因表現分析 68 4.7 菸草轉殖株莖部組織之免疫螢光標定及細胞壁組成分分析 69 4.7.1 菸草轉殖株莖部組織免疫螢光標定分析 69 4.7.2 菸草轉殖株木質部細胞壁組成分分析 71 第五章、 結論 73 第六章、 參考文獻 75 | |
| dc.language.iso | zh-TW | |
| dc.subject | 灰楊 | zh_TW |
| dc.subject | 細胞壁 | zh_TW |
| dc.subject | 類纖維素合成酶 | zh_TW |
| dc.subject | 甘露聚醣 | zh_TW |
| dc.subject | 免疫標定 | zh_TW |
| dc.subject | 木質部成熟 | zh_TW |
| dc.subject | Immunolabeling | en |
| dc.subject | Populus trichocarpa | en |
| dc.subject | Cell walls | en |
| dc.subject | Mannan | en |
| dc.subject | Xylem maturation | en |
| dc.subject | Cellulose synthase-like (CSL) | en |
| dc.title | 野生型灰楊及PtiCSLA1增量轉殖株之細胞壁特性分析 | zh_TW |
| dc.title | Cell Wall Characteristics of Wild-type Populus trichocarpa and Overexpressed PtiCSLA1 Transgenics | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張上鎮(Shang-Tzen Chang),王升陽(Sheng-Yang Wang),張淑華(Shu-Hwa Chang),劉逸軒(I-Hsuan Liu) | |
| dc.subject.keyword | 灰楊,細胞壁,類纖維素合成酶,甘露聚醣,免疫標定,木質部成熟, | zh_TW |
| dc.subject.keyword | Populus trichocarpa,Cell walls,Cellulose synthase-like (CSL),Mannan,Immunolabeling,Xylem maturation, | en |
| dc.relation.page | 87 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-15 | |
| 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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