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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 洪傳揚 | zh_TW |
dc.contributor.advisor | Chwan-Yang Hong | en |
dc.contributor.author | 吳宗翰 | zh_TW |
dc.contributor.author | Tsung-Han Wu | en |
dc.date.accessioned | 2023-08-15T16:27:32Z | - |
dc.date.available | 2023-11-09 | - |
dc.date.copyright | 2023-08-15 | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2023-07-31 | - |
dc.identifier.citation | 莊享愷 (2021) 。對水稻矽轉運蛋白Lsi2啟動子進行多重基因編輯以降低基因表現量。國立臺灣大學農業化學研究所碩士論文。台北市。
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88472 | - |
dc.description.abstract | 砷和鎘會透過必需元素或有益元素通道進入水稻穀粒,因此如何減少水稻中砷和鎘的累積是農業上的一大挑戰。水稻根部中,矽轉運蛋白OsLsi2 ( Low Silicon transporter 2,簡稱 OsLsi2 )和磷轉運蛋白OsPT4 ( Phosphate transporter 4,簡稱 OsPT4)分別是水稻根部吸收三價砷 (AsⅢ) 和五價砷 (AsⅤ) 主要通道;而錳轉運蛋白OsNRAMP5 ( Natural Resistance-Associated Macrophage Protein 5 ,簡稱OsNRAMP5) 則是吸收鎘的主要管道。為了抑制水稻對砷和鎘的吸收,本試驗分為兩個部份進行:(1)利用CRISPR/Cas9基因編輯技術,同時對OsPT4和OsNRAMP5兩個基因進行精準突變;(2)從實驗室先前藉由對OsLsi2啟動子進行多重編輯,篩選出多種OsLsi2不同表達程度的水稻進行水耕和土耕的砷吸收試驗,以篩選出三價砷吸收降低,且不影響水稻產量之水稻品系。第一部分試驗中利用tRNA串聯技術同時對 OsPT4和OsNRAMP5基因進行敲除,轉殖後共獲得30個T0世代品系,經定序後在其中2個品系觀察到鹼基剔除,並從其T1世代篩選出了6種不同基因型的osnramp5-ospt4雙重突變體;第二部分幼苗試驗結果顯示,29-28品系不論是砷處理或者砷+矽共處理的條件下,皆能使地上部砷的累積量分別下降27.1%和51.9%,其OsLsi2表現量也較WT下降65%,將此品系種植於砷汙染土壤,穀粒中的砷累積量也較WT降低30.9%。未來將進行osnramp5-ospt4雙重突變體的砷和鎘的吸收試驗,並希望能透過上述兩種突變體雜交,創造出可同時降低砷和鎘累積的水稻品系。 | zh_TW |
dc.description.abstract | Arsenic and cadmium can enter rice grains through essential element or benefit element channels. Therefore, reducing the accumulation of these substances in rice poses a major challenge in agriculture. In rice roots, the silicon transporter OsLsi2 (Low Silicon transporter 2, OsLsi2) serves as the main channel for absorbing trivalent arsenic (AsⅢ), while the phosphorus transporter OsPT4 (Phosphate transporter 4, OsPT4) facilitates the uptake of pentavalent arsenic (AsⅤ). Additionally, the manganese transporter OsNRAMP5 (Natural Resistance-Associated Macrophage Protein 5, OsNRAMP5) is responsible for the absorption of cadmium. This experiment aimed to inhibit the uptake of arsenic and cadmium in rice and was divided into two parts. Firstly, CRISPR/Cas9 gene editing technology was used to precisely mutate the two genes, OsPT4 and OsNRAMP5, simultaneously. Secondly, the OsLsi2 promoter underwent multiple edits to create a variety of rice plants with different expression levels of OsLsi2. These plants were then screened for arsenic uptake in hydroponic and As-contaminated soil. The goal was to identify rice lines that can reduce the uptake of trivaent arsenic without affecting rice yield. In the first part of the experiment, a total of 30 transgenic lines were obtained in the T0 generation, and after sequencing, base deletion was observed in 2 of these lines. In the T1 generation, 6 different genotypes of osnramp5-ospt4 double mutants were screened. In the second part, the line 29-28 showed promising results. It exhibited a reduction of arsenic accumulation in its shoots by 27.1% and 51.9% under arsenic treatment or arsenic+silicon treatment conditions, respectively. When the line was planted in arsenic-contaminated soil, the expression of OsLsi2 was higher compared to the wild type (WT), resulting in a 30.9% reduction in arsenic accumulation in the grain compared to WT. In future experiments, the osnramp5-ospt4 double mutant will undergo arsenic and cadmium absorption tests. The ultimate goal is to crossbreed the above mutants to create a rice line capable of simultaneously reducing the accumulation of both arsenic and cadmium. | en |
dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-08-15T16:27:32Z No. of bitstreams: 0 | en |
dc.description.provenance | Made available in DSpace on 2023-08-15T16:27:32Z (GMT). No. of bitstreams: 0 | en |
dc.description.tableofcontents | 目錄
口試委員會審定書 i 中文摘要 ii Abstract iii 縮寫字對照表 v 目錄 vii 表目錄 xi 圖目錄 xii 附錄目錄 xiii 壹、前人研究 1 一、環境中的砷和鎘 1 1.1.1砷和鎘的來源及危害 1 1.1.2 土壤中的砷物種 1 1.1.3土壤中的鎘物種 1 二、砷和鎘的吸收和轉運 2 1.2.1 AsV 的吸收和轉運 2 1.2.2 As(Ⅲ)的吸收和轉運 2 1.2.3 有機砷的吸收和轉運 3 1.2.4 Cd的吸收和轉運 3 三、水稻中砷和鎘的解毒機制 4 四、過去藉由操作種植環境降低水稻砷和鎘累積之策略 5 1.4.1改變土壤pH和添加鐵錳氧化物資材 5 1.4.2 灌溉條件 5 1.4.3奈米材料的添加 5 五、操作水稻基因表達降低水稻砷和鎘的累積之策略 6 1.5.1 調控水稻中砷吸收之轉運蛋白基因表達 6 1.5.2 提高水稻中砷解毒之轉運蛋白基因表達 7 1.5.3 調控水稻中鎘吸收之轉運蛋白基因表達 7 1.5.4 提高水稻中鎘解毒之轉運蛋白基因表達 7 六、針對啟動子基因編輯調控基因表現 8 七、基因編輯技術概述 9 7.1.1 CRISPR/Cas9基因編輯技術 9 7.1.2 雙股DNA修補機制 10 八、多重基因編輯 (Multiplex gene editing) 11 九、研究室過去有關Lsi2基因之研究 11 貳、實驗目的 13 參、材料與方法 14 一、試驗材料 14 3.1.1植物材料和生長條件 14 3.1.2 水稻癒傷組織誘導 14 二、質體構築 14 3.2.1 CRISPR/Cas9技術中gRNA片段選擇 14 3.2.2 PTG載體設計 15 3.2.3 FokI與BsaI限制酶截切反應 15 3.2.4 限制酶產物之純化 16 3.2.5 T4 DNA 連接酶黏合反應 16 三、大腸桿菌 (E.coli) 質體純化 17 3.3.1 質體之大腸桿菌熱休克轉型 17 3.3.2 大腸桿菌小量培養 17 3.3.3 大腸桿菌質體DNA純化 17 四、農桿菌轉殖 18 3.4.1 農桿菌電穿孔轉型 18 3.4.2 轉型後農桿菌小量培養 18 3.4.3 轉型後農桿菌大量培養 18 3.4.4 農桿菌感染水稻癒傷組織 19 3.4.5 癒傷組織農桿菌之清洗 19 3.4.6 癒傷組織之篩選與再生 19 五、轉殖株基因與編輯結果鑑定 20 3.5.1 DNA萃取 20 3.5.2聚合酶連鎖反應 (PCR) 和DNA片段瓊脂凝膠分析和回收 20 3.5.3 T&A cloning 21 3.5.4菌落篩選及定序 21 六、轉殖株基因表現量分析 22 3.6.1 轉殖株基因表現量分析 22 3.6.2 樣品收集 22 3.6.3 RNA萃取 22 3.6.4 即時定量聚合酶連鎖反應 ( Real-time quantitative PCR ) 23 七、野生型水稻與OsLsi2啟動子編輯水稻砷累積量 23 3.7.1 幼苗砷累積量試驗 23 3.7.2 野生型水稻和OsLsi2轉殖株在砷汙染土壤中農藝性狀和砷累積量分析 23 3.7.3 樣品消化方法 24 八、統計分析方法 24 肆、結果 25 一、pRGEB32-pPT4-pNRAMP5之轉殖效率 25 二、再生水稻品系之編輯樣態測定 25 三、 T1世代轉基因測定 26 四、大片段缺失品系之T1世代編輯結果分析 26 五、ospt4-osnramp5雙重突變體基因表現量分析 27 六、OsLsi2啟動子編輯水稻基因編輯型態及轉基因分析 27 七、OsLsi2啟動子編輯水稻幼苗之砷累積量測定 27 八、OsLsi2啟動子編輯水稻之OsLsi2基因表現量分析 28 九、砷汙染土壤中OsLsi2啟動子編輯水稻農藝性狀分析 28 十、砷汙染土壤中OsLsi2啟動子編輯水稻榖粒砷累積量 28 伍、討論 30 一、ospt4-osnramp5雙重編輯突變體之分析 30 二、OsLsi2啟動子編輯水稻編輯片段對於砷累積量和基因表現量影響 31 5.2.1 啟動子上不同編輯片段對砷累積量之影響 31 5.2.2 啟動子上不同編輯片段對砷逆境下OsLsi2表現量之影響 31 5.2.3 轉錄因子結合位點缺失影響OsLsi2基因表現量 32 三、OsLsi2啟動子編輯水稻之不同世代對於砷累積量影響 33 5.3.1 砷轉運蛋白基因家族之補償 33 5.3.2 OsLsi2啟動子編輯後之極性定位 33 參考文獻 35 | - |
dc.language.iso | zh_TW | - |
dc.title | 透過基因編輯技術降低水稻吸收砷和鎘轉運蛋白之表達 | zh_TW |
dc.title | Attenuation of Arsenic and Cadmium Uptake Transporter Expression in Rice Through Genome Editing Technology | en |
dc.type | Thesis | - |
dc.date.schoolyear | 111-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 蔡育彰;陸重安;葉顓銘;黃文理 | zh_TW |
dc.contributor.oralexamcommittee | Yu-Chang Tsai;Chung-An Lu;Chuan-Ming Yeh;Wen-Lii Huang | en |
dc.subject.keyword | 水稻,OsLsi2,OsPT4,OsNRAMP5,矽轉運蛋白,磷轉運蛋白,錳轉運蛋白,CRISPR/Cas9,砷,鎘, | zh_TW |
dc.subject.keyword | Rice,OsLsi2,OsPT4,OsNRAMP5,silicon transporter,phosphate transporter,manganese transporter,CRISPR/Cas9,arsenic,cadmium, | en |
dc.relation.page | 64 | - |
dc.identifier.doi | 10.6342/NTU202302340 | - |
dc.rights.note | 未授權 | - |
dc.date.accepted | 2023-08-02 | - |
dc.contributor.author-college | 生物資源暨農學院 | - |
dc.contributor.author-dept | 農業化學系 | - |
顯示於系所單位: | 農業化學系 |
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