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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳昭瑩 | |
| dc.contributor.author | Hsian-Wen Yao | en |
| dc.contributor.author | 姚先玟 | zh_TW |
| dc.date.accessioned | 2021-06-12T18:08:56Z | - |
| dc.date.available | 2009-12-21 | |
| dc.date.copyright | 2007-12-21 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-12-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27539 | - |
| dc.description.abstract | 為探討LsGRP1 (Lilium ‘Star Gazer’glycine-rich protein 1)蛋白質在葵百合葉片之表現，將LsGRP1之cDNA構築於大腸桿菌蛋白質表現系統之載體上，進行LsGRP1表現與純化，發現細菌表現之LsGRP1蛋白質在電泳膠體上有偏移及部分C端胺基酸缺失的現象。進一步利用所得之LsGRP1製作多元抗體，得到含有LsGRP1抗體之兔子血清。使用LsGRP1抗體血清偵測，進行經水楊酸處理之葵百合葉片蛋白質萃取條件之測試，結果顯示利用非離子界面活性劑Triton X-100及還原劑硫氫乙醇(2-mercaptoethanol)萃取所得之蛋白質樣品中可偵測到較高量之38.2 kDa蛋白質訊號，而以高溫高鹼性處理及添加聚乙烯聚吡咯烷酮(polyvinylpolypyrrolidone)成分時可以偵測到約66 kDa的蛋白質訊號，又以高溫高鹼性處理所得66 kDa訊號較強。進一歩針對不同處理之葵百合葉片進行蛋白質萃取、電泳分析及偵測，所有處理皆可穩定地偵測到38.2 kDa蛋白質訊號，且於百合灰黴病菌感染之葉片蛋白質樣品中可偵測到最強的訊號，然而僅有在系統性誘導之系統葉蛋白質樣品中可偵測到66 kDa蛋白質訊號，推測LsGRP1可以巨大複合產物或是不同後轉譯修飾形式存在於植物中，於系統性誘導之系統葉中可以66 kDa蛋白質形式存在。此外，利用抗體偵測葵百合原生質體蛋白質表現以及觀察重組螢光蛋白質標記，探討LsGRP1蛋白質於細胞層次的累積位置，結果顯示於受到水楊酸刺激之原生質體細胞內與細胞外所得之蛋白質樣品中皆可偵測到66 kDa的蛋白質訊號，且隨著處理時間原生質體細胞外所得蛋白質訊號有增加的現象；進一步利用共軛焦顯微鏡(confocol)觀察綠色螢光蛋白質(green fluorescent protein, GFP)標記，結果證明具有LsGRP1訊息序列之重組綠色螢光蛋白質累積於細胞膜內側，推測葵百合葉片中之LsGRP1可能存在於細胞外質或細胞壁。 | zh_TW |
| dc.description.abstract | In order to study expression of LsGRP1 protein in Lilum ‘Star Gazer’ leaves, LsGRP1 cDNA was constructed in protein expression vector of Escherichia coli. LsGRP1 expression and purification were carried on for preparation of LsGRP1 polyclonal antibody. Western blot analysis detected LsGRP1 as a protein of 38.2 kDa following extraction with non-ionic detergent Triton X-100 and reductant β-mercaptoethanol. A protein of 66 kDa was detected following extraction with hot water-NaOH and polyvinylpolypyrrolidone. Extracts of Lilum ‘Star Gazer’ leaves in different treatments were stably detected the protein of 38.2 kDa, but the protein of 66 kDa was only detected in the protein samples of systemic leaves, indicating that posttranslational modification(s) or large mixture composition of LsGRP1 might occur. In addition, the protein of 66 kDa could be detected in SA-treated protoplasts, and protein signal changes from the cell to culture supernatant as the time increased. Confocol microscopy study indicated that LsGRP1-GFP fusion protein was not present evenly in the protoplast cytoplasm, but accumulated inside the cell membrane. Thus, LsGRP1 may exist in the extracellular matrix or cell wall in Lilum ‘Star Gazer’ leaves was presumed. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-12T18:08:56Z (GMT). No. of bitstreams: 1 ntu-96-R94633007-1.pdf: 2974636 bytes, checksum: b3d2cec6b3a0eea40cd32cc670ef2a5c (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 目錄
壹、前言 5 貳、前人研究 7 叁、材料方法 12 一、 細菌質體 DNA 之小量製備法 12 二、 DNA 瓊脂精膠體電泳分析 12 三、 引子的設計 12 四、聚合酵素連鎖反應 13 五、限制酵素切割 13 六、DNA片段的回收與純化 13 七、DNA 黏接反應 14 八、大腸桿菌勝任細胞製備 14 九、大腸桿菌細胞轉形 15 十、大腸桿菌轉形株之篩選 15 十一、菌株的保存 15 十二、細菌重組蛋白質之抽取 16 十三、親和性管柱層析與蛋白質定量 16 十四、蛋白質透析、酵素切割與濃縮 17 十五、SDS-聚丙烯醯胺膠體電泳分析 17 十六、LsGRP1 抗原製備 18 十七、利用液相層析質譜儀分析蛋白質序列 19 十八、西方墨點法 19 十九、LsGRP1 重組蛋白質間交互鍵結作用測試 21 二十、植物材料栽植與處理 21 二十一、葵百合葉片蛋白質萃取 22 二十二、葵百合原生質體製備 22 二十三、葵百合葉片原生質體處理 23 二十四、葵百合原生質體轉型與重組螢光蛋白質暫時表現偵測 23 肆、結果 25 一、利用大腸桿菌蛋白質表現系統可得到GST-LsGRP1重組蛋白質 25 二、由重組蛋白質經酵素反應可得到抗原蛋白質以製作兔子血清抗體 25 三、萃取水楊酸處理之葵百合葉片蛋白質測試結果 26 (一)使用蛋白質常用溶液萃取所得之樣品中可偵測到微弱的38.2 kDa蛋白質訊號 26 (二)以適當之界面活性劑與還原劑萃取所得之樣品中可得較強的38.2 kDa蛋白質訊號 27 (三)NaCl、高溫-高鹼、PVPP及蔗糖萃取葵百合葉片蛋白質可得66 kDa蛋白質訊號 28 四、萃取撲殺熱處理及病原菌接種之葵百合系統葉片蛋白質可得到與水楊酸處理之葵百合葉片蛋白質相同的蛋白質訊號 29 五、受到百合灰黴病菌感染之葵百合葉片可偵測到較強的蛋白質訊號但無66 kDa蛋白質訊號 30 六、誘導處理之葵百合系統葉再接種病原菌之蛋白質分析得到與病原菌接種之葵百合接種葉相似的結果 31 七、不同處理的葵百合葉片中可偵測到38.2 kDa及66 kDa兩種不同形式之LsGRP1蛋白質 31 八、LsGRP1蛋白質間無法利用tyrosine、H2O2與HRP進行交互鍵結 32 九、葵百合原生質體蛋白質分析顯示偵測到之蛋白質可能自細胞內移動至細胞外 33 十、重組螢光蛋白質表現於葵百合原生質體細胞內側 33 伍、討論 35 一、利用大腸桿菌重組蛋白質表現系統得到兩種蛋白質產物 35 二、LsGRP1可能以巨大複合產物或是不同修飾型式存在於植物中 35 三、LsGRP1無法利用tyrosine、H2O2與HRP發生自身交互鍵結 38 四、LsGRP1可能於表現後被傳送至原生質體細胞之外 39 陸、圖表集 41 表一、供試菌株及質體 42 表二、引子對序列 43 表三、植物蛋白質萃取溶液成分(一) 44 表四、植物蛋白質萃取溶液成分(二) 45 表五、植物蛋白質萃取溶液成分(三) 46 表六、經前處理或預先接種之葵百合接種葉使用之植物蛋白質萃取液成分 47 圖一、GST-LsGRP1 重組蛋白質功能區組成及質體構築 48 圖二、GST-LsGRP1核酸序列及重組蛋白質之預測胺基酸序列 49 圖三、E. coli XL1-Blue(pGEX-GL8)及E. coli XL1-Blue(pGEX-6p-1)粗抽蛋白質電泳及西方墨點分析 51 圖四、GST-LsGRP1 重組蛋白質純化電泳分析 52 圖五、純化蛋白質LC-MS/MS分析 53 圖六、水楊酸處理之葵百合葉片萃取蛋白質西方墨點分析(一) 54 圖七、水楊酸處理之葵百合葉片萃取蛋白質西方墨點分析(二) 55 圖八、水楊酸處理之葵百合葉片萃取蛋白質西方墨點分析(三) 56 圖九、未處理之葵百合葉片萃取蛋白質西方墨點分析 57 圖十、化學物質處理與病原菌接種之葵百合系統葉萃取蛋白質之西方墨點分析(一) 58 圖十一、化學物質處理與病原菌接種之葵百合系統葉萃取蛋白質之西方墨點分析(二) 59 圖十二、百合灰黴病菌接種葵百合之接種葉與系統葉萃取蛋白質之西方墨點分析 60 圖十三、經前處理或預先接種之葵百合接種葉萃取蛋白質之西方墨點分析 61 圖十四、LsGRP1 蛋白質間交互鍵結試驗 62 圖十五、葵百合原生質體蛋白質表現之西方墨點分析 63 圖十六、葵百合原生質體轉形後8小時之重組螢光蛋白質表現 64 圖十七、葵百合原生質體轉形後16小時之重組螢光蛋白質表現 65 圖十八、葵百合原生質體轉形後24小時之重組螢光蛋白質表現 66 圖十九、葵百合原生質體轉形後8、16及24小時之重組螢光蛋白質表現 67 柒、參考文獻 68 捌、附錄 75 圖一、GFP重組蛋白質功能區組成 76 | |
| dc.language.iso | zh-TW | |
| dc.title | 葵百合LsGRP1蛋白質於葉片中之誘導表現及定位研究 | zh_TW |
| dc.title | Study on induced expression and localization of LsGRP1 in the leaves of Lilium cv. Star Grazer | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 沈偉強,鄭秋萍,張麗冠,賴爾? | |
| dc.subject.keyword | 葵百合,LsGRP1,多元抗體,葵百合原生質體,綠色螢光蛋白質, | zh_TW |
| dc.subject.keyword | Lilum ‘Star Gazer’,LsGRP1,polyclonal antibody,transient expression,protoplast,GFP fusion protein,confocol microscopy, | en |
| dc.relation.page | 74 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-12-04 | |
| 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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