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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳昭瑩(Chao-Ying Chen) | |
| dc.contributor.author | Che-An Chen | en |
| dc.contributor.author | 陳則安 | zh_TW |
| dc.date.accessioned | 2021-06-16T09:28:48Z | - |
| dc.date.available | 2019-06-12 | |
| dc.date.copyright | 2017-06-12 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-03-30 | |
| dc.identifier.citation | 1. 李敏郎。2000。百合灰黴病之藥劑管理。農政與農情 101:96。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59582 | - |
| dc.description.abstract | 百合受到灰黴病菌 (Botrytis elliptica) 感染後，由防禦相關基因 LsGRP1 所編碼之第二型富含甘胺酸蛋白表現量會提高；基於兩種植物第二型富含甘胺酸蛋白 (菸草之 cdiGRP、阿拉伯芥之 AtGRP-3) 會藉由蛋白–蛋白交互作用活化植物防禦反應，因此 LsGRP1 很可能利用類似的機制介導百合的防禦反應。本研究利用免疫共沉澱法得到數個 LsGRP1 可能的交互作用蛋白，再利用液相色譜串聯質譜儀鑑定其身分，結果顯示這些蛋白參與葉綠體電子傳遞鏈與卡爾文循環。由於這些蛋白在光合作用中扮演重要的角色，推測百合之光照情形會影響 LsGRP1 的表現或是功能。將百合葉片避光處理後，LsGRP1 的累積量會下降，且會在光照後得到補償。此外於百合原生質體中觀察 LsGRP1 之分布，顯示 LsGRP1 會累積在葉綠體，這些結果顯示 LsGRP1 介導的防禦反應與光合作用相關。此外，利用病毒基因靜默系統於百合植株上靜默 LsGRP1 與其假定交互作用蛋白 Rubisco small subunit (RbcS) 之基因，接種試驗顯示 LsGRP1 及 RbcS 基因靜默植株對灰黴病的抗性皆降低。接續測定正常百合與基因靜默植株，於灰黴病菌接種前後光合作用速率與葉綠體相關基因表現之變化，結果顯示灰黴病菌所導致的光合作用效率下降與氣孔關閉有關，而 LsGRP1 與 RbcS 會影響 Photosystem I reaction center subunit N 與 Photosystem II subunit O-2 之基因表現；由於此二基因編碼之蛋白在光合作用反應中扮演重要的角色，推測 LsGRP1 可能會與葉綠體蛋白交互作用共同調控百合的生理生化反應。本研究嘗試闡述 LsGRP1 與其他百合蛋白間的交互作用關係，以及此交互作用關係對於百合抗病性的影響，以促進對於植物第二型富含甘胺酸蛋白參與植物防禦反應機制之瞭解。 | zh_TW |
| dc.description.abstract | The defense-related gene LsGRP1 encoding a plant class II glycine-rich protein (GRP) exhibits an increased level of expression in Lilium spp. after being infected by Botrytis elliptica, the fungal pathogen of lily leaf blight. Referred to two characterized plant class II GRPs (i.e., tobacco cdiGRP and Arabidopsis AtGRP-3) which activate plant defense via protein-protein interactions, LsGRP1 is proposed to implement a similar mechanism. In this study, a number of putative LsGRP1-interacting lily proteins taking a part in electron transport chain and Calvin cycle were identified using co-immunoprecipitation and liquid chromatography-electrospray ionization-tandem mass spectrometry. Due to these putative LsGRP1-interacting proteins playing roles in photosynthesis process, light exposure was proposed affecting the expression and/or function of LsGRP1. Preliminary assay showed that dark treatment could decrease LsGRP1 content which re-built up after light exposure. Fluorescent microscopic examination indicated that LsGRP1 present in the chloroplasts of lily protoplasts. These findings revealed that LsGRP1-mediated defense in lily would be photosynthesis-related. While using virus-induced gene silencing (VIGS) to analyze the functions of LsGRP1 and putative LsGRP1-interacting protein, Rubisco small subunit (RbcS), silenced LsGRP1 and RbcS conducted a more susceptibility to B. elliptica. Changes of photosynthesis rate and chloroplast-related gene expressions before and after fungal inoculation were examined. The results showed that a decrease of photosynthesis rate caused by B. elliptica was related to stomatal closure, and LsGRP1 and RbcS involved in the gene expression of Photosystem I reaction center subunit N and Photosystem II subunit O-2 which playing importants roles in photosynthesis. Thus, LsGRP1 interacting with chloroplast proteins to regulate the physiological and biochemical responses of lily was proposed. This study will elaborate the knowledge on the interactions between LsGRP1 and other lily proteins, and the following effects on disease resistance to get better understanding on the defensive mechanism of plant class II GRPs. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T09:28:48Z (GMT). No. of bitstreams: 1 ntu-106-R03633016-1.pdf: 5762208 bytes, checksum: 05879a7c6ceadc33ba067202dccfa1ed (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 壹、中文摘要 I
貳、英文摘要 II 參、前言 1 肆、前人研究 3 一、百合 3 二、百合灰黴病 4 三、葵百合之水楊酸誘導性防禦相關蛋白 LsGRP1 5 四、植物富含甘胺酸蛋白 (Glycine rich proteins, GRPs) 6 五、植物第二型富含甘胺酸蛋白之交互作用蛋白 10 六、葉綠體介導的植物防禦反應 10 伍、材料與方法 13 一、試驗植物之栽培 13 二、試驗菌株之培養、保存與接種方法 13 1. 菌株培養 13 2. 菌株保存 13 3. 接種源製備與接種方法 13 三、利用大腸桿菌表現 LsGRP1 融合蛋白 14 1. 大腸桿菌表現蛋白菌株之建構與保存 14 2. 誘導融合蛋白表現與胞內蛋白粗萃取 14 3. 蛋白質純化與透析 15 4. 蛋白質定量 16 四、百合蛋白質粗萃取 16 五、免疫共沉澱法 17 六、以十二烷基硫酸鈉聚丙烯醯胺膠體電泳分離與偵測蛋白 17 1. Glycine 與 Tricine 十二烷基硫酸鈉聚丙烯醯胺膠體電泳 17 2. 考馬斯藍染色法 19 3. 銀染法 19 4. 西方墨點法 20 七、高效液相層析電灑法串聯式質譜儀分析 21 八、LsGRP1 交互作用蛋白基因之選殖 21 九、建構病毒誘導基因靜默之百合植株 22 十、水楊酸澆灌處理與病原菌侵入情形之觀察 23 1. 水楊酸澆灌處理 23 2. 以錐蟲藍染色觀察百合葉片中真菌族群 23 3. 以二氨基聯苯胺呈色法觀察過氧化氫分布 23 4. 以苯胺藍染色觀察癒傷葡聚糖分布 24 十一、百合植株之基因表現量分析 24 十二、於百合原生質體觀察 EGFP 標定之 LsGRP1 蛋白的次細胞定位 25 1. 百合原生質體製備 25 2. 由聚乙二醇介導表現載體之轉形 25 十三、光合作用速率測定 26 陸、結果 27 一、釣取 LsGRP1 之交互作用蛋白 27 二、鑑定 LsGRP1 的交互作用蛋白 28 三、百合葉片避光處理會降低 LsGRP1 之累積量∕含量 28 四、LsGRP1 分布於葵百合原生質體葉綠體內 28 五、病毒誘導 LsGRP1 與 RbcS 基因靜默之百合植株對灰黴病菌感病性上升 29 六、水楊酸處理會降低百合胞內二氧化碳濃度、蒸散作用強度及二氧化碳同化速率 30 七、LsGRP1 可能參與水楊酸預處理改變灰黴病菌接種介導之葵百合胞內二氧化碳濃度動態變化的過程 30 八、水楊酸處理之百合植株蒸散作用強度及二氧化碳同化速率在灰黴病菌接種後變化幅度較小 31 九、處理水楊酸與表現 LsGRP1 及 RbcS 基因會抑制光合作用相關之 PsIN 及 OEE2 基因的表現 32 十、接種灰黴病菌會誘導 LsGRP1 及 RbcS 表現 32 十一、病毒誘導 LsGRP1 及 RbcS 基因靜默百合之 PsIN、OEE2 於灰黴病菌接種後表現量大幅下降 33 柒、討論 35 捌、參考文獻 43 玖、圖表集 54 表一、引子對序列 55 表二、預測之百合 LsGRP1 交互作用蛋白液相層析串聯式質譜儀分析結果 56 圖一、利用免疫共沉澱法釣取百合中可能與 LsGRP1 交互作用之蛋白 57 圖二、以考馬斯藍染色法偵測灰黴病菌感染後增強表現之百合蛋白 58 圖三、以免疫共沉澱釣取及銀染法偵測可能與 LsGRP1 交互作用之百合蛋白 59 圖四、灰黴病菌感染後增強表現之百合蛋白 Ls37 序列分析 61 圖五、假定與 LsGRP1 交互作用之百合蛋白 LsIP10 序列分析 62 圖六、假定與 LsGRP1 交互作用之百合蛋白 LsIP19 序列分析 64 圖七、三個 RbcS 重疊群之序列分析及其 qPCR 產物之熔融曲線 67 圖八、LsGRP1 假定交互作用蛋白之 cDNA 選殖與病毒誘導基因靜默載體構築 68 圖九、光照影響 LsGRP1 於百合葉片中的表現 69 圖十、葵百合原生質體轉形後 24 小時之重組 LsGRP1 螢光蛋白次細胞分布 70 圖十一、經病毒介導基因靜默之百合植株接種灰黴病菌後之葉部病徵 72 圖十二、經病毒介導基因靜默之百合植株接種灰黴病菌後之葉表菌絲纏聚情形 74 圖十三、經病毒介導基因靜默之百合植株於接種前之胞間二氧化碳濃度、蒸散作用強度以及二氧化碳同化速率 75 圖十四、經病毒介導基因靜默之百合植株接種灰黴病菌後之胞間二氧化碳濃度 77 圖十五、經病毒介導基因靜默之百合植株接種灰黴病菌後之蒸散作用強度 79 圖十六、經病毒介導基因靜默之百合植株接種灰黴病菌後之二氧化碳同化速率 81 圖十七、百合植株之 LsGRP1、RbcS、PsIN、OEE2 基因相對表現量 83 圖十八、經病毒介導基因靜默之百合植株之 LsGRP1、RbcS、PsIN、OEE2 基因相對表現量 84 圖十九、經病毒介導基因靜默之百合植株於接種灰黴病菌後之 LsGRP1、RbcS、PsIN 及 OEE2 基因相對表現量 87 拾、附錄 88 附圖一、從過表現 EGFP-LsGRP1 之阿拉伯芥中釣取可能與 LsGRP1 交互作用之蛋白 89 附圖二、正常百合及基因靜默空載體對照組百合之 LsGRP1 基因相對表現量 90 | |
| dc.language.iso | zh-TW | |
| dc.title | 百合防禦相關蛋白 LsGRP1 參與水楊酸誘導抗病性及影響光合作用蛋白 | zh_TW |
| dc.title | Defense-related LsGRP1 of lily involves in salicylic acid-induced disease resistance and affects photosynthetic proteins | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃秀珍(Hsiou-Cheng Huang),鄭秋萍(Chiu-Ping Cheng),黃祥恩(Hsiang-En Huang) | |
| dc.subject.keyword | LsGRP1,百合灰黴病,植物第二型富含甘胺酸蛋白,蛋白–蛋白交互作用,光合作用, | zh_TW |
| dc.subject.keyword | LsGRP1,Botrytis elliptica,plant class II glycine-rich protein,protein-protein interaction,photosynthesis, | en |
| dc.relation.page | 91 | |
| dc.identifier.doi | 10.6342/NTU201700734 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-03-30 | |
| 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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