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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林長平 | |
| dc.contributor.author | Yi-Ting Su | en |
| dc.contributor.author | 蘇意婷 | zh_TW |
| dc.date.accessioned | 2021-06-15T07:09:43Z | - |
| dc.date.available | 2013-10-31 | |
| dc.date.copyright | 2010-10-31 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-10-20 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48710 | - |
| dc.description.abstract | 植物菌質體感日日春染後會造成花器發育不正常，包含花瓣褪色、花器綠化、葉片化等及枝條增生、葉片變小、生長遲緩、簇葉、植株萎凋及黃化等病徵。其中造成花器異常的現象即為植物菌質體病害的一種特殊現象。本論文之研究目的即在於探討植物菌質體感染之後植物花器病徵造成的原因以及對植物菌質體生長之意義。研究指出，花器官的發育是由 A class (AP1 及 AP2 ), B class (AP3 及 PI), C class (AG) 及 E class (SEP) 四群基因的交互作用所共同調控的。而花色的生合成則仰賴 chalcone synthase 基因及 chalcone isomerase 基因的作用。因此在本研究中即利用同步聚合酵素連鎖反應之相對定量方法，分析日日春葉片黃化病植物菌質體及花生簇葉病植物菌質體罹病日日春，於花形變化三階段之花形決定基因 B class AP3 及 PI、 C class AG 及 E class SEP3 與花色決定基因 chalcone synthase 基因 (CHS) 及 chalcone isomerase 基因 (CHI) 等各基因表現量之變化。結果顯示 B class 基因中的 AP3 基因的表現量，在兩種植物菌質體感染之花器中皆隨著病徵嚴重程度的增加而逐漸下降，PI 基因於花生簇葉病感染之日日春中有階段性下降的趨勢且下降的幅度較大，但在日日春葉片黃化病感染之日日春中，並無階段性的改變且基因表現的下降幅度較小。C class基因 AG 的表現量，在兩種植物菌質體感染之花器中皆未隨著花形變化嚴重程度之增加而下降。E class 基因 SEP3 基因為花形決定各測定基因中表現量下降最多者，且在花生簇葉病感染之日日春中會隨著病徵嚴重程度之增加而下降，而 SEP3 基因在日日春葉片黃化病感染之日日春中，於第一階段花形變化之花器中表現量最低。花色決定基因當中 CHS 基因的表現量下降程度較 CHI 基因下降程度大。另外，本研究亦利用同步聚合酵素連鎖反應中的絕對定量方法，分析植物菌質體罹病日日春之花器及其花器周圍葉片之菌量，試圖找出植物菌質體與病徵發展的相關性。依罹病日日春花形變化三階段之花器及其周圍葉片菌量之定量結果，顯示在兩種植物菌質體感染之日日春當中，罹病花器菌質體菌量皆隨著花器病徵的嚴重程度的增加而累積量明顯上升，在受花生簇葉病植物菌質體感染之第三階段花形變化之花器中，其菌量累積量高於週圍葉片之菌量累積量達五倍之多，而在受日日春葉片黃化病植物菌質體感染之第三階段花形變化之花器中，其菌量累積量亦高於週圍葉片之菌量累積量達三倍之多。由結果得知，日日春受植物菌質體感染後，花器形態及花色的改變與花形及花色決定基因表現量之下降有關，且植物菌質體之感染造成日日春花器形態之改變，有利於植物菌質體在罹病植株中族群量之增加。 | zh_TW |
| dc.description.abstract | Phytoplasmas infected periwinkles exhibit flower malformation, including petal discoloration, virescence, phyllody, witches’ broom, and other severe symptoms such as leaf yellowing, and stem proliferation. The flower malformation is a very unique symptom in phytoplasma-infected plants. Therefore, this research aims to elucidate how and why phytoplasma cause the special symptom. Because flower organ identity is regulated by A-class (AP1 and AP2), B-class (AP3 and PI), C-class (AG) and-E class gene (SEP) and flower pigment synthesis requires chalcone synthase (CHS) and chalcone isomerase (CHI), four floral organ identity genes, AP3, PI, AG and SEP3 and two pigment synthesis genes, CHS and CHI were analyzed using relative real time RT-PCR to compare the expression levels of those genes in three floral malformation stages of periwinkle infected with periwinkle leaf yellowing phytoplama (PLY phytoplasma) or peanut witches’ broom phytoplasma (PnWB phytoplasma). Expression of all floral organ identity genes and pigment synthesis genes was down-regulated. Suppression of AP3 gene followed the severity of floral malformation stages in both phytoplasma infected periwinkles. The suppression of PI gene was more severe in PnWB phytoplasma infected periwinkles than that in PLY phytoplasma infected plants. Trend of this suppression in PnWB phytoplasma infected periwinkles was similar to that of AP3 suppression. Suppression of AG gene did not follow with the severity of floral malformation stages in both phytoplasma infected periwinkles. SEP3 gene showed the most significant suppression among examined floral organ identity genes in malformed flowers. Suppression of SEP3 also followed the severity of floral malformation in PnWB infected periwinkle ; however, did not exhibit the same correlation with severity in PLY infected periwinkles. Supression of both pigment synthesis genes, CHS and CHI, was close in stage 2 and stage 3 in both phytoplasma infected periwinkles. The suppression of CHI gene is less severe than that of CHS gene. Phytoplasma titers of the defined stages were also determined in flowers and their surrounding leaves using absolute quantitative real time PCR. Following the severity of floral malformation, phytoplasma accumulation increased especially in malformed flowers. At the stage three, the phytoplasma titer was around 5 fold higher in the stage 3 malformed leaf-like flowers than that in leaves in PnWB phytoplasma infected periwinkles, and was more than three fold higher in PLY phytoplasma infected plants. These results indicate that the phyllody and virescence caused by phytoplasma infection are correlated with the down-regulation of floral organ identity genes and pigment synthesis genes in periwinkle, and also suggest that the flower to leaf conversion caused by phytoplasma infection significantly promote phytoplasma accumulation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T07:09:43Z (GMT). No. of bitstreams: 1 ntu-99-R97633016-1.pdf: 3285443 bytes, checksum: 3e865bb42ae3edcf8c603795bddb683a (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 論文口試委員審定書……………………………………………………………...…II
致謝……………………………………………………………………………….….Ⅲ 中文摘要………………………………………………………………………….…..V 英文摘要……………………………………………………………………………..VI 壹、 前言………………………………………………………………………………1 貳、 前人研究…………………………………………………………………………3 一、 植物菌質體之發現與其植物病理學………………………………………3 (一) 植物菌質體之發現……………………………………………………3 (二) 植物菌質體之植物病理學……………………………………………5 二、 植物菌質體之分群…………………………………………………………6 三、 植物菌質體之生物特性與其分子生物學上之研究………………...…….8 四、 花形決定基因之研究………………………………………………….….10 五、 花色決定基因之研究………………………………………………….….12 六、 植物菌質體罹病植物生理變化之研究……………………………….….12 (一) 植物荷爾蒙之相關研究………………………………………….….12 (二) 醣類代謝之相關研究………………………………………………..14 (三) 花器病徵之相關研究………………………………………………..15 (四) 花色之相關研究……………………………………………………..16 參、 材料與方法……………………………………………………………………..17 一、 研究材料…………………………………………………………………..17 (一) 試驗植物來源與繁殖………………………………………………..17 (二) 罹病日日春之花形變化三階段……………………………………..18 (三) 花生簇葉病植物菌質體罹病日日春之花器與植物菌質體菌量之關係……………………………………………………………………..18 二、 罹病日日春之花器形態…………………………………………………..19 (一) 解剖顯微鏡觀察……………………………………………………..19 (二) 石蠟切片觀察………………………………………………………..19 1. 石蠟包埋及切片…………………………………………………..19 2. 脫蠟及染色………………………………………………………..20 三、 健康與受植物菌質體感染之植物全 DNA 及 RNA 之純化………….20 (一) 大量抽取植物全 DNA……………………………………………...20 (二) 微量抽取植物全 DNA……………………………………………...21 (三) 健康及罹病日日春全 RNA 之純化……………………………..…22 四、 花形與花色決定基因之選殖…………………………………………..…23 (一) 第一股 cDNA 合成…………………………………………………24 (二) 聚合酵素連鎖反應 (PCR) 引子對之設計…………………………24 (三) 聚合酵素連鎖反應…………………………………………………..26 (四) 聚合酵素連鎖反應產物之純化與選殖…………………………..…26 1. 聚合酵素連鎖反應產物之膠體萃取 (gel extraction)…………...27 2. 聚合酵素連鎖反應產物之選殖…………………………………..27 (1) 黏合作用 (ligation)……………………………………………27 (2) 轉形作用 (transformation)…………………………………….28 (五) 聚合酵素連鎖反應轉形株之特性分析……………………………..28 1. 以菌落聚合酵素連鎖反應 (colony PCR) 分析轉形株之選殖片段…………………………………………………………..………28 2. 轉形株選殖片段之核酸定序與序列分析…………………….....29 五、 花形與花色決定基因3’ 端序之選殖………………………………….30 (一) 第一股 cDNA 合成…………………………………………………30 (二) 基因特異性引子 (gene specific primer) 之設計……………..……30 (三) 聚合酵素連鎖反應…………………………………………………..31 (四) 聚合酵素連鎖反應產物之純化選殖與序列分析…………….…….31 六、 以南方氏雜配反應 (Southern hybridization) 分析日日春花形、花色決定基因之套組數 (copy number)……………………………………………31 (一) 花形與花色決定基因探針之製備…………………………………..32 1. 核酸探針片段序列之增幅與選殖………………………………..32 (1) 核酸探針片段引子對之設計………………………………….32 (2) 聚合酵素連鎖反應…………………………………………….32 (3) 聚合酵素連鎖反應產物之選殖與序列分析………………….32 2. 核酸探針之標識 (labelling)………………………………...……33 (1) 重組質體DNA之純化……………………………………..….33 (2) 以聚合酵素連鎖反應標識核酸探針………………………….34 (二) 南方氏轉漬 (Southern blot) 及雜配反應 (hybridization)..………..35 1. 健康日日春花器全 DNA 之核酸限制酵素酵解………….……35 2. 南方氏轉漬. ………………………………………………………35 3. 雜配及呈色反應…………………………………………………..36 七、 以同步聚合酵素連鎖反應 (real-time PCR) 之相對定量測定罹病日日春花形變化三階段之花形與花色決定基因之mRNA表現量……………37 (一) 同步聚合酵素連鎖反應引子對之設計……………………………..37 (二) 以相對定量方式測定基因之mRNA表現量………………………38 1. 樣本之收集……………………………………………..…………38 2. 同步聚合酵素連鎖反應 (real-time PCR)……………..…………38 3. 基因表現量之計算………………………………………..………39 八、 以同步聚合酵素連鎖反應 (real-time PCR) 之絕對定量方式測定罹病日日春之花器與葉片帶菌量………………………………………..………40 (一) 同步聚合酵素連鎖反應引子對之設計………………………..……40 (二) 同步聚合酵素連鎖反應標準曲線 (standard curve) 之建構………41 1. 同步聚合酵素連鎖反應標準濃度DNA模板之製備…………....41 (1) 日日春葉片黃化病植物菌質體 amp基因之標準濃度人工DNA模板 (artificial DNA template) 之製備………………...41 (2) 花生簇葉病植物菌質體 imp基因之標準濃度人工DNA模板 (artificial DNA template) 之製備……………………………..42 (3) 健康日日春標準濃度DNA模板之製備……………………...43 2. 同步聚合酵素連鎖反應 (real-time PCR) ……………………….43 3. 標準曲線之建構…………………………………………………..44 (三) 以同步聚合酵素連鎖反應進行罹病日日春花形變化三階段之花器與周圍葉片菌量之定量……………………………………………45 1. 樣本之收集………………………………………………………..45 2. 同步聚合酵素連鎖反應 (real-time PCR)………………………..45 3. 罹病日日春菌量之計算…………………………………………..46 (四) 以同步聚合酵素連鎖反應進行罹病日日春花器與菌質體菌量關係之定量………………………………………………………………..46 1. 樣本之收集………………………………………………………..46 2. 同步聚合酵素連鎖反應 (real-time PCR)………………………..47 3. 罹病日日春菌量之計算…………………………………………..47 肆、 結果…………………………………………………………………………......48 一、 受植物菌質體感染之日日春花器形態之轉變…………………………..48 二、 花形與花色決定基因之分析……………………………………………..48 (一) 花形決定基因之分析………………………………………………..48 1. APETALATA 3 (AP3) 基因之分析………………………………..48 2. PISTILLATA (PI) 基因之分析……………………………………49 3. AGAMOUS (AG) 基因之分析……………………………………50 4. SEPTALLATA (SEP) 基因之分析………………...………………50 (二) 花色決定基因之分析………………………………………………..51 1. chalcone synthase (CHS) 基因之分析…………….……………..51 2. chalcone isomerase (CHI) 基因之分析…………………………..51 三、 植物菌質體感染造成的花形轉變與變化之相關性分析………………..52 (一) 花形與花色決定基因同步聚合酵素連鎖反應之引子對專一性測定………………………………………………………….………….52 (二) 植物菌質體感染造成的花形轉變與變化之相關性分析.………….54 四、 罹病日日春花形變化三階段花器及其周圍葉片之帶菌量……….……56 (一) 同步聚合酵素連鎖反應之引子對專一性測定…………..…………56 (二) 同步聚合酵素連鎖反應標準曲線之建構…………………………..57 1. 以 PLY-ampf2 / PLY-ampr2 引子對建構日日春葉片黃化病植物菌質體菌量之標準曲線…………………………..………………57 2. 以 PnWB-impf1 / PnWB-impr1 引子對建構花生簇葉病植物菌質體菌量之標準曲線…………………………..…………………58 3. 以UBQ-1252F/ UBQ-1392R 引子對建構日日春DNA質量之標準曲線…………………………..…………………………………59 (三) 以同步聚合酵素連鎖反應進行罹病日日春花形變化三階段花器及其周圍葉片菌量之定量……………………………………………..59 五、 花與花生簇葉病植物菌質體菌量增殖之關係…………………………..60 伍、 討論……………………………………………………………………………..62 陸、 參考文獻………………………………………………………………………..68 柒、 圖表……………………………………………………………………………..79 | |
| dc.language.iso | zh-TW | |
| dc.title | 植物菌質體罹病日日春花器葉片化、綠化之病徵發展及花形、花色決定基因之變化與植物菌質體菌量之關係 | zh_TW |
| dc.title | The Correlation of Symptom Development in Phyllody and Virescence with Gene Expressions in Floral Organ Identity and Pigment Synthesis and with Phytoplasma Accumulation in Phytoplasma-infected Catharanthus roseus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 陳仁治 | |
| dc.contributor.oralexamcommittee | 曾國欽,張碧芳,洪挺軒 | |
| dc.subject.keyword | 花器綠化,花器葉片化,花形相關決定基因,花色相關決定基因,同步聚合酵素連鎖反應, | zh_TW |
| dc.subject.keyword | floral organ identity genes,phyllody,pigment synthesis genes,real-time PCR,virescence, | en |
| dc.relation.page | 132 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-10-20 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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