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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉瑞芬 | |
| dc.contributor.author | Siao-Huei Yi | en |
| dc.contributor.author | 易筱蕙 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:31:52Z | - |
| dc.date.available | 2017-08-18 | |
| dc.date.copyright | 2012-08-18 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64143 | - |
| dc.description.abstract | 亞磷酸處理番茄後能誘導植物產生防禦反應，並有效抵抗疫病菌 (Phytophthora parasitica)侵染，為瞭解亞磷酸啟動抗病反應的相關機制，本實驗室先前以生物晶片進行基因表現分析，結果發現亞磷酸處理可誘導許多pathogenesis-related genes的表現，其中包括一未知功能之putative PR protein (命名為NPA-induced pathogenesis-related protein a, NIPRa)，本研究之目的在探討NIPRa的功能與特性。除了NIPRa，番茄另含有NIPRb基因，在基因組中位於NIPRa下游，與NIPRa胺基酸序列之相似度高達87.5 %。此外，於其他物種也可發現NIPRa同源性序列，範圍遍及動物、細菌、藻類、卵菌及真菌，但功能都尚未明瞭。經亞磷酸處理植物或以疫病菌及青枯病菌 (Ralstonia solanacearum)感染植物後，NIPRa表現量都會明顯上升；水楊酸及乙烯也會誘導NIPRa表現。利用PVX病毒載體在番茄植株中系統性表現NIPRa後，可使番茄植株對疫病菌及青枯病菌的耐病力都略為提升，若以TRV-induced gene silencing靜默番茄NIPRa之基因表現後，再接種病原菌，也可觀察到番茄植株對於疫病菌及青枯病菌之感病度都較為增加，顯示NIPRa參與在番茄抵抗這兩種病原菌的防禦反應中。為探討哪些序列參與NIPRa基因之轉錄調控，我們以GUS為報導基因，應用agroinfiltration技術在菸草葉片進行NIPRa之啟動子分析，結果顯示轉譯起始點上游248 bp至599 bp之序列區間可能包含重要的基因表現調控序列。此外，以細菌大量表現NIPRa重組蛋白，並進行圓二色光譜分析的結果顯示，其結構中近70 % 為α-helix，且於pH 7-8時，二級結構最穩定，Tm值則為60 oC。由於純化之重組蛋白呈現綠色，且加入EDTA會影響其構型，推測NIPRa重組蛋白極可能嵌合著金屬離子；進一步進行ICP-MS分析發現，NIPRa重組蛋白所嵌合之離子可能為鎳離子。本實驗為首度針對NIPRa蛋白之功能與特性所進行的研究，其在植物細胞之作用方式及在植物生理所扮演的角色尚待進一步的釐清。 | zh_TW |
| dc.description.abstract | Phosphonate-based fungicides such as neutralized phosphorous acid (NPA) are known to induce plant resistance against many diseases, including those caused by Phytophthora. To investigate the mechanism underlying NPA-induced resistance, we previously performed a microarray analysis and found that a variety of defense genes were induced in response to NPA treatment on tomato plants. Among them, one gene (named NPA-induced pathogenesis-related protein a, NIPRa), which showed homology to a putative pathogenesis-related (PR) gene in barley, is significantly induced but functionally unknown. Hence, the aim of this study is to uncover the characteristics of NIPRa. Analysis by semi-quantitative reverse transcriptase-PCR indicated that expression of NIPRa was induced when plants were challenged with either P. parasitica or the bacteria wilt pathogen Ralstonia solanacearum. NIPRa was up-regulated by salicylic acid and ethylene treatment as well. To test whether NIPRa contributes to plant resistance against pathogens, we overexpressed NIPRa by PVX agroinfection, and then challenged the plants with either P. parasitica or R. solanacearum. Plants overexpressing NIPRa showed higher tolerance to infection by these pathogens. In contrast, down-regulation of NIPRa by TRV-induced gene silencing increased plant susceptibility to pathogen infection. In addition, the promoter of NIPRa gene was analyzed by agroinfiltration of tobacco leaves, using GUS as a reporter, to define sequence elements that are required for P. parasiticaresponse. The result showed that a 351-bp region between 248 and 599 bp upstream of NIPRa translation start site was essential for induction by P. parasitica. Furthermore, the NIPRa recombinant protein purified from E. coli showed green color and tended to form dimers to polymers when analyzed by gel filtration. Analysis by ICP-MS indicated that it is most likely a metalloprotein associated with nickel ion. These results suggested that NIPRa may represent a novel category of PR protein, yet its function needs further investigation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:31:52Z (GMT). No. of bitstreams: 1 ntu-101-R98633017-1.pdf: 3096624 bytes, checksum: 12529e3e9915208f65abbec1aff8529f (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 中文摘要 i
英文摘要 ii 壹、前言 1 1、 番茄簡介 1 2、 植物防禦反應 (Plant defense response) 1 3、 Pathogenesis-related Proteins (PRs) 3 4、 亞磷酸 (Neutralized Phosphorous Acid)誘發抗病反應，並誘導產生PRs 7 5、 研究動機及策略 8 貳、材料與方法 9 1、 供試植株及菌種來源 9 2、 亞磷酸處理試驗及病原菌接種 10 3、 多序列比對分析及蛋白質功能結構預測 11 4、 NIPRa及NIPRb之RNA表現分析 12 5、 番茄內大量表現NIPRa (Gene Overexpression) 14 6、 番茄內進行NIPRa 基因靜默化 (Gene Silencing) 16 7、 NIPRa localization 17 8、 NIPRa蛋白純化及特性分析 19 9、 啟動子分析 (Promoter analysis) 21 叁、結果 24 1、 亞磷酸處理可誘導Pathogenesis-related genes (PR genes)的表現 24 2、 番茄NIPRa及NIPRb 的選殖及鑑定 24 3、 番茄基因NIPRa及NIPRb與其他同源性基因之親緣樹分析 25 4、 NIPRa及NIPRb之基因表現情形 25 5、 利用PVX病毒載體在番茄大量表現NIPRa可提高植株對疫病菌及青枯病菌的耐病性 26 6、 利用病毒載體TRV在番茄內進行NIPRa基因靜默 (NIPRa gene silencing)使番茄植株對疫病菌及青枯病菌較感病 27 7、 NIPRa在細胞內的表現位置 28 8、 菸草植株處理亞磷酸及疫病菌後NIPRa啟動子活性分析 29 9、 利用3D結構預測NIPRa之功能 30 10、NIPRa蛋白之表現與純化 30 11、 以圓二色光譜(circular dichroism, CD)分析NIPRa重組蛋白 32 12、 以ICP-MS (Inductively-Coupled Plasma Mass Spectrometry)分析NIPRa重組蛋白所帶有的金屬離子 33 肆、討論 34 1、 鑑定NIPRa為PRs 34 2、 番茄內NIPRa與NIPRb 34 3、 NIPRa之同源性基因分析 35 4、 NIPRa對疫病菌及青枯病菌的耐病性 36 5、 NIPRa啟動子分析 38 6、 NIPRa蛋白質結構功能 40 7、 結語 41 伍、參考文獻..................................................................................................................42 陸、附表………………………………………………………………………………..49 柒、附圖………………………………………………………………………………..54 八、補充資料…………………………………………………………………………..73 | |
| dc.language.iso | zh-TW | |
| dc.subject | 疫病菌 | zh_TW |
| dc.subject | 亞磷酸 | zh_TW |
| dc.subject | 番茄 | zh_TW |
| dc.subject | pathogenesis-related genes | zh_TW |
| dc.subject | 青枯病 | zh_TW |
| dc.subject | neutralized phosphorous acid (NPA) | en |
| dc.subject | pathogenesis-related (PR) gene | en |
| dc.subject | promoter analysis | en |
| dc.subject | Phytophthora parasitica | en |
| dc.subject | Ralstonia solanacearum | en |
| dc.title | 探討番茄pathogenesis-related protein NIPRa之特性與功能 | zh_TW |
| dc.title | Characterization of NIPRa, a pathogenesis-related protein from Solanum lycopersicum | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 賴爾?,鄭秋萍,林乃君,徐駿森 | |
| dc.subject.keyword | pathogenesis-related genes,番茄,亞磷酸,疫病菌,青枯病, | zh_TW |
| dc.subject.keyword | neutralized phosphorous acid (NPA),pathogenesis-related (PR) gene,promoter analysis,Phytophthora parasitica,Ralstonia solanacearum, | en |
| dc.relation.page | 77 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-15 | |
| 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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