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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 常玉強(Yuh-Chyang Charng) | |
| dc.contributor.author | Chung-Yu Chen | en |
| dc.contributor.author | 陳中宇 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:58:47Z | - |
| dc.date.available | 2021-07-10T21:58:47Z | - |
| dc.date.copyright | 2021-03-08 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-01-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77374 | - |
| dc.description.abstract | 基因編輯技術可以精準地在目標 DNA產生期望之插入(insertion)、刪除(deletion)以及取代(substitution)突變 ，而CRISPR/Cas9為當今基因編輯最有潛力的工具，但先前研究多僅利用Cas9蛋白產生雙股DNA斷裂，進而選擇修復錯誤之個體並觀察基因剔除(knock-out)產生的結果。最近發展的先導編輯(prime editing)技術可利用連接反轉錄酶的Cas9融合蛋白直接於指定位置創造期望突變。 本研究利用先導編輯使水稻內生EPSPS基因產生新功能(gain-of-function)，透過取代其中兩個胺基酸使水稻由對嘉磷塞敏感型轉為抵抗型，兩點的突變分別為第172個glycine突變為alanine及第215個glycine為aspartic acid。透過農桿菌轉殖法，於台農67號水稻轉入辨識突變位置的pegRNA、兩位點各自的nicking spacer及nCas9-RT基因。最終成功獲得6株轉殖系，其中3株更在其中G215突變位置成功偵測到期望突變的發生 。 | zh_TW |
| dc.description.abstract | Gene editing techniques can achieve specific and precise gene modification by DNA insertion, deletion or substitution. CRISPR/Cas9 system has been proven as the most powerful tool for genome modifications, while most efforts results in “knock-out” of a gene because of DNA double strands break by Cas9. Recently, “Prime editing” was developed that directly writes desired codon into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase. In this study, PE (prime editing) technique was used to create a “gain-of-function mutant” EPSPS gene of rice for glyphosate resistant by replacing two amino acids. The replacing indicates the codon for the glycine residue at position 172 was changed to encode an alanine residue and the codon for the glycine at position 215 was changed to encode an aspartic acid residue. The specific pegRNA as well as nicking RNA constructs were synthesized, together with nCas9-RT gene, and transformed into rice TNG67. Successful transgenic rice plants were confirmed and three of them have been detected expected point mutation. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:58:47Z (GMT). No. of bitstreams: 1 U0001-1901202112335600.pdf: 2902852 bytes, checksum: 9a6b182eb3bb291865144ac87e022eb1 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 口試委員會審定書…………………………………………………… i 中文摘要…………………………………………………………… ii 英文摘要………………………………………………………… iii 目錄………………………………………………………………… iv 表目錄…………………………………………………………… vi 圖目錄………………………………………………………… vii 壹. 前言……………………………………………………………… 1 一. 莽草酸途徑 (shikimic acid pathway)…………………… 1 二. EPSPS基因與嘉磷塞 (glyphosate) 抗性育種…………… 2 三. 基因編輯技術的發展及其優缺點……………………… 3 四. Knock-in的重要性……………………………… 6 五. 透過prime editing技術編輯水稻EPSPS基因……… 7 貳. 材料方法……………………………………………………… 8 一. 大腸桿菌勝任細胞製備…………………………………… 8 二. 質粒的大腸桿菌轉形………………………………………… 8 三. 大腸桿菌質粒DNA的萃取與純化………………………… 8 四. 水稻農桿菌轉殖……………………………………………… 9 五. 水稻DNA萃取…………………………………………… 11 六. 構築設計……………………………………………… 11 七. PCR檢測轉殖株…………………………………… 13 八. 嘉磷塞濃度試驗……………………………………… 13 參. 結果…………………………………………………… 14 一. pPEEP構築設計……………………………………… 14 二. 水稻轉殖與篩選T0轉殖株………………………………… 15 三. 檢測T0轉殖株EPSPS基因發生之鹼基置換…………… 15 四. 水稻癒傷組織嘉磷塞抗性測試…………………………… 15 肆. 討論……………………………………………………………… 16 伍. 參考文獻………………………………………………………… 19 陸. 附錄 - 溶液與培養基………………………………………… 25 | |
| dc.language.iso | zh-TW | |
| dc.subject | Knock-in | zh_TW |
| dc.subject | 先導編輯 | zh_TW |
| dc.subject | 嘉磷塞 | zh_TW |
| dc.subject | EPSPS | zh_TW |
| dc.subject | CRISPR | zh_TW |
| dc.subject | 精準育種 | zh_TW |
| dc.subject | Prime editing | en |
| dc.subject | Knock-in | en |
| dc.subject | Precise breeding | en |
| dc.subject | CRISPR | en |
| dc.subject | EPSPS | en |
| dc.subject | Glyphosate | en |
| dc.title | 應用先導編輯修飾水稻EPSPS基因 | zh_TW |
| dc.title | Modification of Rice EPSPS Gene by Prime Editing | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 109-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 杜 鎮 (Jenn Tu),洪傳揚(Chwan-Yang Hong) | |
| dc.subject.keyword | 先導編輯,嘉磷塞,EPSPS,CRISPR,精準育種,Knock-in, | zh_TW |
| dc.subject.keyword | Prime editing,Glyphosate,EPSPS,CRISPR,Precise breeding,Knock-in, | en |
| dc.relation.page | 40 | |
| dc.identifier.doi | 10.6342/NTU202100090 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2021-01-19 | |
| 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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