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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王愛玉 | |
dc.contributor.author | Chih-Yu Chen | en |
dc.contributor.author | 陳志祐 | zh_TW |
dc.date.accessioned | 2021-06-15T03:54:42Z | - |
dc.date.available | 2010-07-05 | |
dc.date.copyright | 2010-07-05 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-06-29 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44777 | - |
dc.description.abstract | 在快速生長的竹筍中,細胞壁多醣類的生合成相當旺盛。本論文藉由篩選cDNA庫及RT-PCR,共選殖出10株纖維素合成酶 (cellulose synthase, CesA) 之cDNA (BoCesA1~BoCesA10),其中7株具有完整的譯讀序列 (open reading frame),分別可轉譯出1061∼1080個胺基酸(分子量介於 120.77∼122.24 kDa)。 BoCesA1-BoCesA8這8株cDNA彼此在核苷酸序列及胺基酸序列分別有62%-97% 及 67-98% 的同質性,且其胺基酸序列上皆具有植物CesA 序列的特徵,包括:zinc-binding domain、植物CesA所特有的高度變異區域 (HVRI 及HVRII)、八個預測的貫膜部位,及具有高度保守性的UDP-glucose 鍵結部位 (D及DxD motif) 及催化部位 (D 及QxxRW motif) 之可溶性部位。以演化樹分析發現,BoCesA1-BoCesA8 分別屬於三群 (clades),其中BoCesA1, BoCesA2, BoCesA3及BoCesA8屬於VI-A次群,BoCesA4, BoCesA5屬於V-A次群,而BoCesA6及BoCesA7則屬於I-A次群。
由Southern分析的結果推測BoCesA2、BoCesA5、BoCesA6、BoCesA7在綠竹的染色體中有2-3個拷貝。以quantitative real-time PCR分析此4種BoCesA基因在不同綠竹組織及於試管中培育的綠竹多芽體中之基因表現圖譜,顯示此4種BoCesA基因普遍在綠竹各組織器官中表現,但受到差異性調控。另外,BoCesA基因的表現會受到α-naphthaleneacetic acid的負調控與thidiazuron差異性的影響。由in-situ RT-PCR分析結果顯示出BoCesA2、BoCesA5、BoCesA6及BoCesA7在未出土的竹筍基部及節間是普遍存在,而在綠竹側枝的節間中則是主要表現在維管束中央部位,其中BoCesA2及BoCesA6 mRNA的分佈具空間特異性。藉由TAIL-PCR選殖得BoCesA2 及BoCesA5之5’端序列,以CpGisland2 及CpGplot 程式分析,可在其上搜尋得可能的 core promoter element (CpG island)。此外,在BoCesA2 之轉譯起始點上游序列中,也存在一些類似於參與荷爾蒙調節、維管束專一性表現及光反應有關的cis elements。 另以GST pull-down、yeast two-hybrid 及免疫共沈澱等實驗探討BoCesA是否會與催化UDP-glucose 生成的酵素間具有交互作用。結果顯示, BoCesA7與蔗糖合成酶 (sucrose synthase, SuS) 間具交互作用,但BoCesA7 與UDP-glucose焦磷解酶 (UDP-glucose pyrophosphorylase, UGPase) 間則否。此外,也發現在積貯組織中SuS 與UGPase具交互作用。目前正以雙螢光互補分析法 (biomolecular fluorescence complementation assay) 進一步驗證BoCesA7、BoSuS及BoUGPase 蛋白質間的交互作用。 | zh_TW |
dc.description.abstract | Synthesis of cell wall polysaccharides is highly active in rapidly growing bamboo shoots. Ten cDNAs (BoCesA1-BoCesA10) encoding cellulose synthase (CesA) were cloned from Bambusa oldhamii by screening a bamboo shoot cDNA library and/or by RT-PCR. Among them, seven clones possessed complete open reading frame encoding polypeptides between 1061 and 1080 amino acid residues with relative molecular masses between 120.77 and 122.24 KDa, respectively. BoCesA1-BoCesA8 shared 62-97% identity at the nucleotide level and 67-98% identity at the amino acid level with each other. The deduced amino acid sequences of BoCesA1-BoCesA5, BoCesA7 and BoCesA8 contained the conserved features of plant CesA, including a zinc-binding domain, two plant-specific HVRs (HVR-I and HVR-II), eight potential transmembrane domains, and a soluble cytosolic domain with highly conserved D, DxD and D residues and a QxxRW motif. Phylogenetic analysis of CesAs from different plants suggested that the eight BoCesAs belonged to three clades: BoCesA1, BoCesA2, BoCesA3 and BoCesA8 belonged to clade VI-A; BoCesA4 and BoCesA5 belonged to clade V-A and BoCesA6 and BoCesA7 belonged to clade I-A. Southern blot analysis revealed that BoCesA2, BoCesA5, BoCesA6 and BoCesA7 had 2-3 copies in bamboo genome. The expression profiles of the four BoCesA genes in the different bamboo tissue and the in-vitro cultured bamboo multiple shoots were analyzed by quantitative real-time PCR. The four genes were widely expressed in various bamboo organs and bamboo multiple shoots, but were differentially regulated. They were down-regulated by α-naphthaleneacetic acid and differentially affected by thidiazuron in the multiple shoots. In situ RT-PCR analyses demonstrated that BoCesA2, BoCesA5, BoCesA6 and BoCesA7 mRNAs were all present throughout the base and internode of etiolated shoots that emerged from pseudorhizomes. Expression of the four genes in the internodes of branch shoots that emerged from the nodes of mature bamboo culms was predominantly detected in the center of vascular bundles, and spatial-specific expression was observed with BoCesA2 and BoCesA6. 5'-flanking sequences of BoCesA2 and BoCesA5 cloned by TAIL-PCR contained a core promoter element, CpG Island, which was predicted with the CpGisland2 or CpGplot programs. Several elements with similarity to cis-elements that have been known to be involved in hormone responses, vascular-specific expression and light response were detected in the sequence upstream the translation initiation site of BoCesA2.
GST pull-down assay, yeast two-hybrid system and co-immunoprecipitation were used to investigate whether BoCesA interacts with sucrose synthase (SuS) and UDP-glucose pyrophosphorylase (UGPase). SuS and UGPase are both involved in catalyzing the formation of UDP-glucose for polysaccharide synthesis. The results revealed that protein-protein interactions occurred between BoCesA7 and SuS but not between BoCesA7 and UGPases. In addition, the interactions between SuS and UGPase, which occurred in sink tissues of bamboo, were also observed. Further analyses of the interactions between BoCesA7, SuS and UGPase by bimolecular fluorescence complementation assay are in progress. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T03:54:42Z (GMT). No. of bitstreams: 1 ntu-99-D92b47206-1.pdf: 27718309 bytes, checksum: 3153624bc33256549d61f0a2ee052e08 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 目錄………………………………………………………………………Ⅰ
縮寫表…………………………………………………………………………Ⅱ 中文摘要…………………………………………………………………Ⅲ 英文摘要…………………………………………………………………Ⅳ 第一章 緒論 1. 纖維素 (cellulose)......................................1 1.1 纖維素的結構..........................................1 1.2 原核生物中的纖維素的合成與相關基因....................2 1.3 植物纖維素合成的部位及酵素............................3 1.4 高等植物中的纖維素合成酶基因及序列特性................4 1.5 纖維素合成酶之基因....................................6 1.6 阿拉伯芥中AtCesA 的研究...............................6 1.7阿拉伯芥中CSC中CesA的組成形式..........................8 1.8影響阿拉伯芥植株細胞壁生合成non-CesA突變株之研究.......9 1.9 其它植物中CesA的研究.................................12 1.10高等植物中纖維素合成的可能機制.......................14 2. 竹與竹筍的簡介.........................................14 2.1 竹與竹筍生長的特色...................................16 3. 本論文之研究緣起.......................................16 4. 本論文的研究主題.......................................17 4.1綠竹筍CesA cDNA 之選殖與序列分析......................17 4.2 BoCesA基因啟動子序列選殖與可能cis-element分析........18 4.3 BoCesA基因輿綠竹不同生長階段下的基因表現分析.........18 4.4 BoCesA蛋白質分析.....................................18 4.5 BoCesA與SuS及UGPase是否具蛋白質交互作用分析..........18 第二章 材料與方法 1. 實驗材料: 1.1 植物材料: 1.1.1 綠竹(Bambusa oldhamii)...........................20 1.1.2 綠竹多芽體 (bamboo multiple shoots)............20 1.1.3 菸草(Nicotiana benthamiana)懸浮細胞............20 1.2載體..................................................21 1.3菌種..................................................23 2. 實驗藥品..............................................24 3. 實驗儀器..............................................24 4. 實驗方法: 4.1 綠竹cDNA庫之篩選...................................25 4.1.1 噬菌體價數測定 (titering)......................25 4.1.2 cDNA庫之放大及轉印.............................25 4.1.3 random priming法製備放射性CesA探針.............26 4.1.4 預雜合與雜合反應...............................26 4.1.5具正反應噬菌體之單離............................26 4.2 正反應cDNA株之檢定.................................27 4.2.1 胞內剪切 (in vivo excision)....................27 4.2.2 質體DNA之小量分離法............................27 4.2.3南方轉印法......................................28 4.2.4 雜合反應.......................................28 4.3 cDNA尾端快速增殖法(RACE).......................................28 4.3.1 第一股cDNA 的合成..............................29 4.3.2 cDNA的純化.....................................30 4.3.3 cDNA 5'端加上oligo-dC tailing..................30 4.3.4 dC-tailed cDNA 的 PCR 增殖.....................30 4.3.5 Nested PCR amplification.......................31 4.4 以RT-PCR 選殖 BoCesA6 及 BoCesA7的5'-端............31 4.5 以GeneGOEing 選殖具有完整ORF之BoCesA7 cDNA.........33 4.6 BoCesA定序及序列分析...............................34 4.7 BoCesA基因拷貝數之分析.............................35 4.7.1 竹筍染色體DNA之抽取............................35 4.7.2 PCR法製備放射性標幟探針........................36 4.8 以TAIL-PCR法進行BoCesA 基因啟動子序列選殖..........37 4.9 即時反轉錄酶-聚合酶連鎖反應 (real-time RT-PCR)....40 4.9.1 綠竹 total RNA抽取.............................41 4.9.2 變性甲醛瓊脂糖膠體電泳.........................42 4.9.3 反轉錄 (reverse transcription) 反應............43 4.9.4 PCR............................................44 4.9.5 Internal control 基因表現穩定性測試............45 4.10 纖維素及木質素含量分析............................45 4.10.1 纖維素含量分析................................45 4.10.2 木質素含量分析................................46 4.11原位反轉錄增殖法 (in situ RT-PCR)..................46 4.11.1 植物組織切片..................................47 4.11.2 Azure blue染色................................48 4.11.3 RT-PCR........................................48 4.11.4 設計適用於in-situ RT-PCR的核酸引子............49 4.11.5 免疫組織呈色分析..............................52 4.11.6 TBO染色.......................................53 4.12 BoCesA-HVR重組蛋白質表現現........................53 4.12.1表現質體的建構.................................53 4.12.2 重組蛋白質表現條件探討........................54 4.12.3 His-tag::BoCesAHVRI及HVRII之內涵體純化........55 4.12.4 IMAC純化......................................55 4.12.5 SDS膠體電泳...................................56 4.12.6 Coomassie Brilliant Blue 染色法...............56 4.12.7 Western轉印...................................56 4.12.8 免疫分析呈色法................................57 4.12.9 Zinc/imidazole 膠體負染色法...................57 4.12.10 In-gel digestion與ESI-MS-MS 分析.............57 4.13 BoCesA 專一性抗體製備.............................58 4.13.1 BoCesA多源性抗體製備..........................58 4.13.2 Monospecific抗體製備..........................58 4.13.3抗體效價測試...................................59 4.14 綠竹筍原生質膜分劃與脂質筏 (lipid rafts)純化......59 4.14.1 綠竹筍原生質膜之純化..........................59 4.14.2 綠竹筍脂質筏的純化............................60 4.15 GST pull-down 分析................................60 4.15.1 GST-Tag表現質體的建構.........................61 4.15.2 GST-Tag重組蛋白質的表現.......................62 4.15.3 以Glutathione Sepharose純化重組蛋白質.........62 4.15.4 BoSuS1及BoSuS2重組蛋白質表現與純化............62 4.15.5 水稻懸浮細胞及綠竹筍蛋白質粗抽液之製備........63 4.15.6 以GST pull-down分析...........................63 4.16 BoUGPase選殖與重組蛋白質表現......................63 4.16.1 BoUGPase cDNA 之選殖..........................63 4.16.2 BoUGPase重組蛋白質的表現......................64 4.17 Yeast two-hybrid分析.............................65 4.17.1表現質體之建構.................................65 4.17.2 酵母菌之轉形..................................68 4.17.3 Yeast mating..................................68 4.18 雙分子螢光互補(BiFc)分析.........................69 4.18.1 菸草懸浮細胞之原生質體製備....................69 4.18.2 作用於雙分子螢光互補系統之表現質體建構........70 4.18.3 菸草原生質體轉形..............................74 4.19 綠色螢光蛋白質標定(GFP-targeting)................74 4.19.1 GFP-targeting表現質體建構.....................74 4.20免疫共沉澱分析....................................76 4.20.1 以RSuS1 多源抗體進行免疫沉澱分析..............76 4.20.2 以純化的BoUGPase、BoSuS進行免疫沉澱分析.......77 4.20.3以BoUGPase 多源抗體進行免疫沉澱................77 第三章 結果與討論 1. BoCesA cDNA之選殖....................................78 1.1 BoCesAs正選殖株的鑑定..............................78 1.2 以5'RACE法對BoCesA7的5'端片段進行選殖..............79 1.3 以RT-PCR對BoCesA6及BoCesA7的5'端片段進行選殖.......79 1.4 BoCesA1-BoCesA10 cDNA 序列及其同質性分析...........81 1.5 不同植物CesA與綠竹BoCesA之演化分析.................81 2. BoCesA cDNA胺基酸序列之結構特徵......................82 2.1 Zinc-finger domain 的預測..........................82 2.2 貫膜部位分析.......................................82 2.3 UDP-glucose鍵結區與催化區間的預測..................83 2.4 HVRI及HVRII的預測..................................83 2.5 可能的醣基化修飾位置...............................84 3. 綠竹筍中BoCesA 基因拷貝數之探討......................84 3.1專一性探針之製備....................................84 3.2 BoCesA基因拷貝數分析...............................85 4. BoCesA 之上游5'-端序列選殖...........................85 4.1 BoCesA2及BoCesA5轉譯起始點上游序列分析.............85 5.BoCesA基因在綠竹不同生長時期及不同組織之表現量 5.1 Internal control 表現穩定性分析....................87 5.2 PCR引子對及產物專一性分析..........................88 5.3 在綠竹中不同生長階段與組織中BoCesAs基因表現情形....88 6.纖維素與木質素含量分析 7. Cytokinin與auxin對BoCesA 基因表現的影響..............89 8. 不同BoCesA mRNA 在綠竹組織中的定位...................90 8.1綠竹組織切片中核酸的完整性..........................90 8.2專一性引子的設計....................................91 8.3 In situ RT-PCR反應的差異...........................92 8.3.1 在未出土竹筍中BoCesA mRNA的分佈................92 8.3.2 在綠竹側枝中BoCesA mRNA的分佈..................93 9. 部份重組BoCesA 蛋白質表現及抗體製備..................94 9.1表現BoCesA HVRI及HVRII 重組蛋白質片段...............94 9.2 BoCesA抗體的製備...................................95 9.2.1 以BoCesA HVRI及HVRII作為抗原製備抗體所需.......95 9.2.2 針對BoCesA6及BoCesA7之專一性抗體製備...........96 9.3 以BoCesA 抗體分析綠竹中的BoCesA....................97 9.3.1 BoCesA 蛋白質的穩定性..........................97 9.3.2 探討BoCesA 但白質是否位於原生質膜上的DRMs......98 10. 以GST pull-down探討BoCesA7CR與其他蛋白質間交互作用...99 10.1 GST::BoCesACR重組蛋白質的表現與純化...............99 10.2以水稻懸浮細胞蛋白質粗抽液進行GST pull-down........99 10.3以純化之重组BoSuS及BoUGPase進行GST pull-down......100 11. 以yeast two-hybrid 方式探討BoCesA7CR與其他蛋白質間交互作用.....................................................101 11.1 Prey及bait表現質體self-activation 測試...........101 11.2 以重組bait及prey質體進行yeast two-hybrid 之結果..102 12. 以雙分子螢光互補方式探討BoCesA7與其他蛋白質間的交互作用.......................................................102 13. 以免疫共沉澱分析BoUGPase與BoSuS間交互作用...........103 13.1 以 SuS1 多源性抗體進行免疫沉澱...................103 13.2 以 GPase 多源性抗體進行免疫沉澱分析..............103 13.3 分析SuS與UGPase於細胞內的交互作用................104 第四章 結論與未來展望 4.1結論.............................................106 4.2未來方向 4.2.1參與次級細胞壁生合成BoCesA cDNA選殖與表現分析.106 4.2.2 BoCesA蛋白質表現與免疫組織分析...............106 4.2.3 BoCesA 重組蛋白質穩定性及可能調控機制探討....107 4.2.4 CSC 探討.....................................107 參考文獻.............................................109-126 表目錄...............................................127-134 圖目錄...............................................135-200 | |
dc.language.iso | zh-TW | |
dc.title | 綠竹中與初級細胞壁生合成相關之纖維素合成酶基因探討 | zh_TW |
dc.title | Analysis of the Cellulose Synthase Genes Associated with Primary Cell Wall Synthesis in Bambusa oldhamii | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 蘇仲卿,宋賢ㄧ,林耀輝,鄭石通,楊健志 | |
dc.subject.keyword | 纖維素合成酶,初級細胞壁,綠竹, | zh_TW |
dc.subject.keyword | cellulose synthase,primary cell wall,bambusa oldhamii, | en |
dc.relation.page | 200 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-06-29 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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