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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張孟基(Men-Chi Chang) | |
| dc.contributor.author | Bo-Yuan Huang | en |
| dc.contributor.author | 黃柏元 | zh_TW |
| dc.date.accessioned | 2021-06-07T18:06:08Z | - |
| dc.date.copyright | 2020-09-17 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-13 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16235 | - |
| dc.description.abstract | 表觀遺傳學 (Epigenetics) 乃透過DNA甲基化程度或組織蛋白的修飾，在不改變基因序列下調控基因表現並影響作物外表型。基因型相同的個體，若具有不同的表觀遺傳修飾或修飾程度差別會產生不一樣的變異，此現象稱『表觀遺傳變異 (Epigenetic variation)』。在遺傳育種中，可以做為另一路徑提供作物外表型變異的新來源。除了透過自然產生、逆境誘導，現今還可利用分子技術創造表觀遺傳變異個體，並根據表觀遺傳修飾位置是否具有專一性區分為標靶或非標靶表觀遺傳修飾 (Targeted or Non-targeted epigenetic modification)。本研究目的主要在探討利用小鼠染色質修飾因子mTRIM28是否能應用於阿拉伯芥，作為發展新的非標靶表觀遺傳修飾工具。若能利用染色質修飾因子mTRIM28作為非標靶表觀遺傳修飾工具，透過隨機影響基因組中表觀遺傳修飾狀態，便可以創造出具有不同變異的族群 (Epi-mutant pool)。為達此目的，首先根據胺基酸序列比對結果，發現阿拉伯芥中並沒有如mTRIM28具有多功能區域的單一蛋白質，而是由具有不同個別相似功能域之諸多蛋白質。進而透過次細胞表現部位結果顯示mTRIM28的核定位訊號可以透過阿拉伯芥importin α1/importin β轉運至細胞核中，接著以異位表現mTRIM28於阿拉伯芥了解mTRIM28是否可透過表觀遺傳修飾影響生長與發育，透過正常或非生物逆境下外表型分析，並以及酵母菌雙雜交系統篩選是否有可能與mTRIM28結合之蛋白質。推測在植物體中可能因為缺乏KRAB-或KRAB-Like鋅指轉錄因子而無法調控基因表現。故若直接以mTRIM28作為非標靶表觀遺傳學修飾工具較不可行。但未來若能利用dCAS9-KRAB解決植物缺乏KRAB-鋅指轉錄因子問題，並發展為標靶表觀遺傳修飾工具。藉由dCAS9-KRAB透過匯集mTRIM28並與阿拉伯芥中組織蛋白修飾酶、染色質重塑蛋白形成異染色質複合物，在不影響基因序列下精準抑制目標基因表現的標靶表觀遺傳修飾系統，將可成為具有發展潛能的新植物育種技術 (New Plant Breeding Technology, NPBT)。 | zh_TW |
| dc.description.abstract | Epigenetics is used for describing the heritable phenotype changes of crops caused by DNA methylation and histone modification without altering the intrinsic DNA sequences, in which phenotypic variation has formed epigenetic diversity for plant breeding. Depending on different extents of epigenetic modification, the individuals can display variation on phenotypic diversity. This represents a new and alternative tool for modern crop improvement. In addition to natural generation and stress induction, nowadays molecular techniques are used to create epigenetic variation, including the target or non-target epigenetic modifications. Based on this, the purpose of this study is to explore whether we can take the chromatin-modifying factor mTRIM28 (Mus musculus Tripartite Motif Containing 28) as a new molecular tool for non-target epigenetic modifications in Arabidopsis. Once the chromatin-modifying factor mTRIM28 can be used as a non-target epigenetic modification tool, an epi-mutant population with epigenetic variation can be created for changing the epigenetic status in the plant genome. Thus, we conducted an amino acid sequence homologous search, there is no single protein with multifunctional domains like mTRIM28 in Arabidopsis. Instead, the genome in Arabidopsis consists of many different proteins with corresponding similar functional domains of mTRIM28. Next, we showed that the nuclear localization signal of TRIM28 can efficiently transport the protein into the nucleus through the Arabidopsis importin α1/importin β system. Furthermore, phenotypic investigation and yeast two-hybrid analysis were performed to explore whether TRIM28 can cause growth and development abnormalities through epigenetic modification, and screen for proteins that are likely to interact with mTRIM28 in Arabidopsis thaliana. As a result, it suggests that Arabidopsis may lack KRAB- or KRAB-Like zinc finger transcription factors； thus it prevents mTRIM28 from silencing gene expression, making TRIM28 non-target epigenetic modification tool is less feasible. However, from my study for future application of mTRIM28 on crop epigenetic breeding, using dCAS9-KRAB/ TRIM28 will be good for targeted epigenetic medication. We expect that this targeted epigenetic modification system holds up the potential for accurately blocking the target gene expression without altering the DNA sequence and represents a possible New Plant Breeding Technology (NPBT) for crop improvement in near future. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T18:06:08Z (GMT). No. of bitstreams: 1 U0001-3007202011444300.pdf: 8091777 bytes, checksum: 32cc465835b3593a7dd8fe38bb05b9de (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 摘要 I Abstract II 縮寫字對照 IV 目錄 VII 第一章 前人研究 1 一、表觀遺傳學調控作物在生長發育及逆境下之反應 2 二、表觀遺傳學機制 3 1.DNA甲基化 3 2.組蛋白修飾 5 三、表觀遺傳學育種 6 1.非標靶表觀遺傳修飾 (Non targeted epigenetic modification) 策略 7 2.標靶表觀遺傳修飾 (Targeted epigenetic modification) 策略 8 四、哺乳動物中TRIM28扮演的生理角色與作用分子機制 10 五、研究目的 11 第三章 材料與方法 14 一、mTRIM28在阿拉伯芥中同源蛋白質搜尋之分析 14 二、試驗材料 14 1. 基因轉殖阿拉伯芥 14 1.1 基因轉殖阿拉伯芥構築與流程 14 1.2 電穿孔法 14 1.3 阿拉伯界基因轉殖-花序感染法 15 1.4 基因轉殖阿拉伯芥種子消毒以及栽培方式 15 1.5 基因轉殖阿拉伯芥篩選同質結合子植株 15 三、阿拉伯芥轉殖株之分子鑑定 16 1.植物基因體DNA抽取 16 2.聚合酶連鎖反應 16 3. RNA抽取與製備 16 4.半定量聚合酶連鎖反應(Semi-quantitative PCR, RT-PCR) 16 5.即時聚合酶連鎖反應 (Real time polymerase chain reaction, qPCR) 17 四、阿拉伯芥外表型調查 17 1.正常處理下外表型調查 17 2.非生物逆境處理下外表型調查 17 五、mTRIM28蛋白質之次細胞表現部位 18 1.短暫表現質粒之構築 18 2.阿拉伯芥原生質體之分離分法 18 3.阿拉伯芥原生質體PEG基因轉殖法 19 4.以共軛焦顯微鏡觀察mTRIM28蛋白質之次細胞表現部位 20 六、阿拉伯芥轉殖株之全基因體表現量分析 20 七、酵母菌雙雜合系統 21 1.pGBKT7- mTRIM28質粒之構築 21 2.勝任酵母菌制備 21 3.勝任酵母菌PEG基因轉殖法 22 4.酵母菌雙雜交系統與篩選 22 4.1 Y2HGold Yeast Strain [pGBKT7- mTRIM28]自我活化測試 22 4.2 酵母菌雙雜交系統 23 八、統計分析方法 24 第四章 結果 25 一、阿拉伯芥中具有mTRIM28相似功能區域蛋白質 25 二、mTRIM28蛋白質之次細胞表現位置 25 三、異位表現mTRIM28基因之阿拉伯芥轉殖株分子鑑定 25 四、異位表現mTRIM28對阿拉伯芥生長發育之影響 26 1. mTRIM28轉殖株之根系調查 26 2. mTRIM28轉殖株開花期調查 26 3. mTRIM28轉殖株營養生長期調查 27 4.乾旱逆境下mTRIM28轉殖株之外表型調查 27 5.高鹽逆境下mTRIM28轉殖株幼苗之外表型調查 27 五、異位表現mTRIM28阿拉伯芥轉殖株之全基因體表現分析 27 六、酵母菌雙雜交系統篩選阿拉伯芥中與mTRIM28交互作用之蛋白質 28 第五章 討論 30 一、阿拉伯芥中具有與mTRIM28相似功能區域蛋白質 30 二、mTRIM28蛋白質可能透過阿拉伯芥importin α1/importin β進入細胞核 32 三、探討mTRIM28應用於非標靶表觀遺傳修飾工具的可行性 34 四、人造KRAB轉錄因子協調mTRIM28於阿拉伯芥發揮表觀遺傳修飾功36 五、人造KRAB轉錄因子/mTRIM28標靶表觀遺傳修飾工具應用於表觀遺傳育種 37 第六章 未來研究 39 一、dCAS9-KRAB載體設計 39 二、PDS3作為標靶表觀遺傳修飾位點以及gRNA設計 39 三、建立dCAS9-KRAB/ mTRIM28系統於阿拉伯芥轉殖株中 40 第七章 結論 41 參考文獻 42 圖目錄 圖1、mTRIM28胺基酸序列比對阿拉伯芥基因組。 54 圖2、mTRIM28蛋白質之次細胞表現部位 55 圖3、異位過量表現mTRIM28阿拉伯芥轉殖株分子鑑定 56 圖4、異位過量表現mTRIM28阿拉伯芥轉殖株外表型調查 57 圖5、異位過量表現mTRIM28阿拉伯芥轉殖株逆境外表型調查 58 圖6、異位過量表現mTRIM28阿拉伯芥轉殖株與野生型差異表現基因分析 59 圖7、酵母菌雙雜交系統鑑定mTRIM28交互作用蛋白 60 圖8、酵母菌雙雜交系統篩選mTRIM28交互作用之蛋白質 61 圖9、mTRIM28轉運進入細胞核預測模型 62 圖10、小鼠與阿拉伯芥中同源基因結構圖 63 圖11、小鼠與阿拉伯芥中TRIM28作用機制圖。 64 圖12、dCAS9-KRAB/mTRIM28標靶表觀遺傳修飾系統於阿拉伯芥中預測模型 65 表目錄 表1、TRIM28胺基酸序列比對阿拉伯芥基因表。 66 表2、小鼠與阿拉伯芥同源基因功能表。 67 附錄 附錄1、小鼠TRIM28蛋白質結構 68 附錄2、TRIM28蛋白在哺乳類動物中作用機制 69 附錄3、mTRIM28序列優化 76 附錄4、高通量分析阿拉伯芥葉面積 77 附錄5、本研究基因轉殖之載體圖表 78 附錄6、上調基因資訊表 79 附錄7、下調基因資訊表 80 附錄8、本研究所使用引子表-1 81 附錄9、本研究所使用引子表-2 82 附錄10、gRNA序列以及位置 83 附錄11、pCAMBIA1305.1和pSMAB704載體 84 附錄12、D-Clone載體 85 | |
| dc.language.iso | zh-TW | |
| dc.subject | NPBT | zh_TW |
| dc.subject | 表觀遺傳育種 | zh_TW |
| dc.subject | 表觀遺傳修飾工具 | zh_TW |
| dc.subject | TRIM28 | zh_TW |
| dc.subject | dCAS9 | zh_TW |
| dc.subject | dCAS9 | en |
| dc.subject | Epigentic breeding | en |
| dc.subject | Epigenetic modification tools | en |
| dc.subject | NPBT | en |
| dc.subject | TRIM28 | en |
| dc.title | 染色質修飾因子mTRIM28應用於阿拉伯芥表觀遺傳育種的可行性探討 | zh_TW |
| dc.title | Exploring the Possible Application of a Chromatin-Modifying Factor mTRIM28 in Epigenetic Breeding of Arabidopsis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 洪傳揚(Chwan-Yang Hong),謝旭亮(Hsu-Liang Hsieh),鄭萬興(Wan-Hsing Cheng),侯新龍(Shin-Lon Ho) | |
| dc.subject.keyword | 表觀遺傳育種,表觀遺傳修飾工具,TRIM28,dCAS9,NPBT, | zh_TW |
| dc.subject.keyword | Epigentic breeding,Epigenetic modification tools,TRIM28,dCAS9,NPBT, | en |
| dc.relation.page | 85 | |
| dc.identifier.doi | 10.6342/NTU202002086 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-14 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農藝學研究所 | zh_TW |
| 顯示於系所單位： | 農藝學系 | |
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