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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃鵬林(Pung-Ling Huang) | |
dc.contributor.author | En-Ning Wang | en |
dc.contributor.author | 王以寧 | zh_TW |
dc.date.accessioned | 2021-06-08T05:16:50Z | - |
dc.date.copyright | 2006-01-27 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-01-23 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24139 | - |
dc.description.abstract | 核醣核酸干擾技術(RNA interference, RNAi)是默化基因的策略之一,由雙股RNA(dsRNA)誘導同源目標基因產物mRNA的降解作用。因此,應用核醣核酸干擾技術可作為植物抗病毒的轉殖策略。本試驗以胡瓜嵌紋病毒(Cucumber mosaic virus, CMV) 外鞘蛋白 ( coat protein, CP)基因作為RNAi抗病策略之目標默化基因。經比對CMV CP DNA序列,分別將保守性序列25 bp共5區和基因全長657 bp進行RNAi質體之構築。其構形為2x 35S CaMV啟動子、反義序列、香蕉ACC氧化酶基因 (1-aminocyclopropane-1-carboxylate oxidase, MAO1) 第一個隱子、順義序列和35S poly A 終結子。經聚乙二醇 (polyethylene glycol, PEG) 轉殖法將CMV CP報導質體分別與各默化質體共轉殖至蝴蝶蘭花瓣原生質體,以Real-time polymerase chain reaction進行CMV CP基因默化分析。其結果顯示,短片段默化質體piCR4和全長默化質體piCAS確實干擾並降低原生質體內CMV CP RNA含量,默化效率為30﹪。另一方面,藉由農桿菌媒介共培養轉殖法,將各默化質體穩定性轉殖至香蕉北蕉 (Musa AAA cv. Pei Chiao) 和邊沁菸草 (Nicotiana benthamiana) 。植株再生後,經GUS活性組織化學染色分析,各轉殖試驗事件獲得為數不等之擬轉殖株。邊沁菸草擬轉殖株DNA經專一性引子對進行PCR檢測,以及南方氏雜交分析,確定為轉殖株,此等植株可供日後接種CMV進行抗病試驗,以進一步瞭解轉殖株對CMV之抗病性。 | zh_TW |
dc.description.abstract | RNA interference (RNAi) in eukaryotic organisms is an effective strategy for gene silencing. It is involved in sequence-specific degradation of targeted RNA by RNA inducing silencing complex. RNAi makes a new strategy possible to create virus-resistance transgenic plants. In this study, we have selected five conserved regions, 25 bp in length, from Cucumber mosaic virus coat protein (CMV CP) nucleotide sequences and the full length coding region (657 bp) for construction of silencing vectors as effector plasmids. The constructs contuined including 2x 35S CaMV promoter, sense sequence, banana 1-aminocyclopropane-1-carboxylate oxidase gene (MAO1) intron 1, antisense sequence and 35S poly A terminator. Reporter plasmid encoding CMV CP was co-transformed with various effector plasmids via polyethylene glycol into protoplasts isolated from Phalaenopsis petals. The total RNA from transformed protoplasts was extracted for real-time polymerase chain reaction assay to identify the silencing efficiency. The CP RNA was reduced by 30% for the fourth homologous region (piCR4) and the full length silencing plasmid (piCAS). On the other hand, the effector plasmids were stably transformed into banana (Musa AAA cv. Pei Chiao) and Nicotiana benthamiana by Agrobacteria-mediated transformation system. Genomic DNA from regenerated transgenic Nicotiana benthamiana were extracted individually, and polymerase chain reaction and Southern analysis were used to confirm the transgenic plantlets. | en |
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dc.description.tableofcontents | 摘要………………………………………………………………………1
Abstract…………………………………………………………………2 壹、前人研究……………………………………………………………8 一、 胡瓜嵌紋病毒 ………………………………………………8 二、 轉錄後基因默化 ……………………………………………8 (一) 轉殖病毒反義基因之抗病機制 ……………………………9 (二) 轉殖順義基因引發PTGS之機制 ……………………………9 (三) CMV之抗病研究 ……………………………………………10 1.轉殖順義外鞘蛋白基因於抗CMV之研究 …………………10 2.轉殖順義複製酶基因於抗CMV之研究 ……………………10 (四) 核糖核酸干擾技術 (RNA interfernce, RNAi)…………11 1.RNAi之基因默化機制………………………………………11 2.植物體內之RNAi訊息移動…………………………………12 3.RNAi轉殖載體之構築………………………………………13 4.應用轉錄後基因默化於植物抗病之優點…………………13 貮、材料與方法 ………………………………………………………14 一. 試驗材料……………………………………………………14 (一) 植物材料……………………………………………………14 1.香蕉癒傷組織………………………………………………14 2.香蕉懸浮培養細胞系………………………………………14 3.菸草組培苗…………………………………………………14 4.蝴蝶蘭花瓣…………………………………………………14 (二) 胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)相關材枓 ………………………………………………………………14 (三) 質體材料……………………………………………………14 1.中間質體……………………………………………………14 2.轉殖質體……………………………………………………14 二. 試驗方法……………………………………………………14 (一) 核糖核酸干擾(RNA interference, RNAi)之載體構築策略 ………………………………………………………………17 (二) 默化CMV CP基因之載體構築………………………………17 1.PCR 引子設計………………………………………………17 2.聚合酶連鎖反應 (polymerase chain reaction)………17 3.限制酶切和DNA片段回收 …………………………………17 4.接合反應(ligation)………………………………………19 5.大腸桿菌勝任細胞(competent cell)之製備……………19 6.大腸桿菌之轉型……………………………………………19 7.大腸桿菌質體DNA小量製備 ………………………………19 8.大腸桿菌質體DNA大量製備 ………………………………20 9.農桿菌勝任細胞(competent cell)之製備………………21 10.農桿菌電穿孔轉型法………………………………………22 11.農桿菌質體DNA小量製備 …………………………………22 (三) 應用農桿菌媒介轉殖法於香蕉懸浮細胞之穩定性基因轉殖 ………………………………………………………………23 (四) 應用農桿菌媒介轉殖法於菸草和邊沁菸草葉片之穩定性基因轉殖 …………………………………………………………………23 (五) 擬轉殖株分析………………………………………………24 1.GUS活性組織化學染色法 …………………………………24 2.植物基因組DNA之抽取 ……………………………………24 3.植物total RNA之抽取(Guanidinium Thiocyanate Method)…………………………………………………………………25 4.植物葉片蛋白質之抽取……………………………………26 5.核酸探針之製備和同位素標定……………………………26 6.南方氏雜交分析……………………………………………27 7.北方雜交分析………………………………………………27 8.間接式酵抗體法(Indirct Enzyme linked immunosorbent assay, In-ELISA) …………………………………28 (六) 植物原生質體分離…………………………………………29 1.菸草葉片原生質體分離……………………………………29 2.蝴蝶蘭花瓣原生質體分離…………………………………30 (七) PEG轉殖法於植物原生質體之暫時性基因轉殖 …………30 (八) 電穿孔轉殖法於植物原生質體之暫時性基因轉殖………30 (九) 轉殖默化載體於植物細胞的默化效果分析………………31 1.原生質體蛋白之抽取和定量………………………………31 2.原生質體RNA之抽取和定量 ………………………………31 3.Poly (A)+ RNA之純化和微定量 …………………………32 4.反轉錄聚合酶連鎖反應 (RT-PCR) ………………………32 5.即時偵測聚合酶連鎖反應(Real-time PCR) ……………33 參、結果 ………………………………………………………………34 一、 默化載體之構築……………………………………………34 (一) 暫時性報導載體之構築……………………………………34 (二) 穩定性轉殖載體之構築……………………………………34 二、 以暫時性基因轉殖分析默化載體之默化效果……………39 (一) 應用PEG轉殖法於菸草原生質體之暫時性基因轉殖和ELISA檢測 ……………………………………………………………………39 (二) 應用PEG轉殖法於蝴蝶蘭花瓣原生質體之暫時性基因轉殖和Real-time PCR分析…………………………………………………42 (三) 菸草葉片和蝴蝶蘭花瓣原生質體電穿孔轉殖法之條件測試 ………………………………………………………………42 三、應用農桿菌媒介轉殖法於菸草和邊沁菸草葉片之穩定性基因轉殖及分析 ………………………………………………………………46 (一) 菸草擬轉殖株之分析………………………………………46 (二) 邊沁菸草擬轉殖株之分析…………………………………53 四、 應用農桿菌媒介轉殖法於香蕉懸浮細胞之穩定性基因轉殖及分析 …………………………………………………………………59 肆、討論 ………………………………………………………………63 一、 香蕉基因轉殖後之體胚再生………………………………63 二、 胡瓜嵌紋病毒外鞘蛋白基因默化質體在原生質體之默化效果分析 …………………………………………………………………64 三、 抑制基因默化的病毒蛋白…………………………………66 伍、參考文獻 …………………………………………………………68 | |
dc.language.iso | zh-TW | |
dc.title | 胡瓜嵌紋病毒外鞘蛋白基因默化載體之構築及分析 | zh_TW |
dc.title | Construction and Analysis of Silencing Plasmids Specific for Cucumber mosaic virus Coat Protein Gene | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-1 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 杜宜殷(Yi-Yin Do) | |
dc.contributor.oralexamcommittee | 許圳塗(Chou Tou Shii),張清安(Chin-An Chang) | |
dc.subject.keyword | 胡瓜嵌紋病毒,核糖核酸干擾技術,基因轉殖,基因默化, | zh_TW |
dc.subject.keyword | Cucumber mosaic virus,RNA interference (RNAi),gene transformation,gene silencing, | en |
dc.relation.page | 76 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2006-01-24 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 園藝學研究所 | zh_TW |
顯示於系所單位: | 園藝暨景觀學系 |
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