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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳昭瑩 | |
dc.contributor.author | Whei-Shin Chang | en |
dc.contributor.author | 張卉欣 | zh_TW |
dc.date.accessioned | 2021-06-13T06:20:02Z | - |
dc.date.available | 2006-01-27 | |
dc.date.copyright | 2006-01-27 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-01-26 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34643 | - |
dc.description.abstract | 本實驗室在研究百合與灰黴病菌交互作用的過程中,分析自葵百合葉片回收之接種液蛋白質及進一步之分子選殖得到lpglu1 cDNA,解碼為一葡聚醣酶(β-1,3-glucanase),分子量為35.34 kDa,pI值為4.14,與植物酸性β-1,3-葡聚醣酶有高相似性。本研究以lpglu1專一性引子對進行PCR,可分別在雜交種鐵炮百合和台灣百合之基因體核酸增幅出大小類似於lpglu1的單一條帶核酸片段,最適增幅溫度各為54-57℃及55℃左右(葵百合lpglu1之最適黏合溫度為57-60℃)。進一步利用大腸桿菌XL1-Blue菌株表現LPGlu1蛋白質,可以pQE 30載體表現去掉訊息胜肽之LPGlu1 (His-lpg),並可偵測到β-1,3-葡聚醣酶活性,7.34 U/mg。另一方面,以反轉錄聚合酶連鎖反應配合南方雜合分析偵測lpglu1在葵百合葉片之表現時間點及表現量,結果顯示以灰黴病菌接種及水楊酸、茉莉酸甲酯、離層酸或乙烯前驅物處理葵百合皆可以誘導lpglu1 mRNA之累積;此外,對葵百合植株施以傷口、低溫及高鹽處理亦皆可誘導lpglu1的表現。 | zh_TW |
dc.description.abstract | In the study of the interaction between Botrytis elliptica and Lilium cv. Star Gazer, analysis of recovery of inoculation fluids and a subsequent cDNA cloning identified the LPGlu1 protein (35.34 kDa, pI 4.14), sharing high similarity to various plant acidic β-1,3-glucanases. Through PCR technique, specific primers to lpglu1 could be used to amplify relevant fragment from L. longiflorum (hybrid) and L. formosanum genome. The appropriate annealing temperature is between 54-57℃ and 55℃, and the annealing temperature of lpglu1 gene in Star Gazer is about 57-60℃. LPGlu1 without signal peptide could be expressed in Escherichia coli XL1-Blue strain by pQE 30 protein expression system. His-lpg displayed β-1,3-glucanase activity of 7.34 U/mg. The expression of lpglu1 in ‘Star Gazer’ leaves were investigated by RT-PCR and Southern hybridization. The results indicated that the accumulation of lpglu1 mRNA could be induced by B. elliptica, salicylic acid, methyl jasmonate, abscisic acid and 1-aminocyclopropane-1-carboxylic acid. In addition, wounding, cold and high salt could induce the expression of lpglu1 gene. | en |
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dc.description.tableofcontents | 目 錄
壹、 中文摘要……………………………………………………………………….1 貳、 英文摘要……………………………………………………………………….2 參、 前言…………………………………………………………………………….3 肆、 前人研究……………………………………………………………………….5 一、 百合………………………………………………………………………...5 二、 植物的系統性誘導抗病…………………………………………………...5 三、 SAR在百合之研究………………………………………………………..7 四、 植物β-1,3-葡聚醣酶與抗病之關係………………………………………7 五、 β-1,3-葡聚醣酶在百合之研究……………………………………….........9 六、 大量表現植物β-1,3-葡聚醣酶之相關研究……………………………...10 七、 β-1,3-葡聚醣酶在高等植物之誘導表現…………………………………12 伍、 材料與方法……………………………………………………………………14 一、 百合植株之栽培……………………………………………………….….14 二、 灰黴病菌接種源之製備..…………………………………………………14 三、 誘導抗病性試驗………..…………………………………………………14 四、 lpglu1序列電腦分析……..……………………………………………….15 五、 專一性引子之設計………………………………………………………...15 六、 百合基因體核酸之抽取…………………………………………………...16 七、 核酸探針之製備…………………………………………………………...16 八、 南方雜合分析……………………………………………………………...17 九、 聚合酵素連鎖反應及其產物檢查…………………………………………18 十、 PCR產物之回收與選殖…………………………………………………..18 十一、 PCR產物與質體核酸之黏合反應……………………………………..18 十二、 大腸桿菌勝任細胞之製備………………………………………………19 十三、 大腸桿菌勝任細胞之轉形………………………………………………19 十四、 轉形大腸桿菌之篩選…………………………………………………...20 十五、 大腸桿菌質體之抽取與限制酵素之切割………………………………20 十六、 核酸定序分析…………………………………………………………..21 十七、 化學、物理與生理逆境處理……………………………………..……21 1. 水楊酸處理………………………………………………………….....21 2. 茉莉酸甲酯處理……………………………………………………….22 3. 乙烯前驅物處理……………………………………………………….22 4. 離層酸處理…………………………………………………………….22 5. 傷口處理……………………………………………………………….23 6. 低溫處理……………………………………………………………….23 7. 高濃度NaCl溶液處理………………………………………………...23 十八、 百合全RNA之抽取……………………………………………………..23 十九、 反轉錄聚合酵素連鎖反應及南方雜合分析…………………………..24 二十、 大腸桿菌轉形株全蛋白質之抽取……………………………………..24 二十一、 轉形株GST-lpg融合蛋白質之純化………………………………...25 二十二、 聚丙烯醯胺膠體電泳分析…………………………………………..25 二十三、 西方轉漬分析………………………………………………………..26 二十四、 β-1,3-葡聚醣酶活性之偵測與分析…………………………………26 1. β-1,3-葡聚醣酶膠體活性染色………………………………………..27 2. 還原糖酵素活性分析………………………………………………...27 陸、 結果…………………………………………………………………………….28 一、 葵百合lpglu1基因之特性分析……………………………………………28 二、 誘導抗病性試驗……………………………………………………………28 三、 葵百合lpglu1基因分析…………………………………………………..29 四、 親和性病原與不親和性病原對葵百合lpglu1基因誘導表現之差異…..29 五、 不同化學誘導物質處理對葵百合lpglu1基因誘導表現之差異………..30 1. 水楊酸處理…………………………………………………………...30 2. 茉莉酸甲酯處理……………………………………………………...31 3. ACC與離層酸處理…………………………………………………...31 六、 不同物理與生理逆境處理對葵百合lpglu1基因誘導表現之差異……..32 1. 傷口處理……………………………………………………………...32 2. 低溫處理與高鹽處理………………………………………………...32 七、 利用大腸桿菌表現LPGlu1之試驗………………………………………32 1. 以pBluescript SK (N-terminal β-galactosidase)表現LPGlu1…..…...32 2. 以pGEX 6p-1 (GST fusion)表現LPGlu1……………………………33 3. 以pQE 30 (6×His-taq)表現LPGlu1………………………………….33 柒、 討論…………………………………………………………………………….34 捌、 參考文獻……………………………………………………………………….40 玖、 圖表集……………………………………………………………………….....50 表一、 LPGlu1 與其他植物葡聚醣酶相似胺基酸序列之比對………………...51 表二、 各轉形株β-1,3-葡聚醣酶活性之偵測………………………………....52 圖一、 lpgl u1核苷酸組成分析………………………………………………….53 圖二、 LPGlu1 蛋白質疏水性分析…………………………………………….54 圖三、 LPGlu1 與其他植物β-1,3-葡聚醣酶胺基酸序列之排並分析………..55 圖四、 誘導抗病性試驗………………………………………………………...57 圖五、 lpglu1 引子設計(引子212、213、214、261 及262)……………………..58 圖六、 lpglu1 cDNA 與基因之序列比對……………………………..…….….59 圖七、 利用lpglu1 專一性引子對對不同品種百合基因體核酸進行PCR 之 電泳圖……..……………………………………………………………60 圖八、 葵百合基因體核酸之南方雜合分析…………………………………..61 圖九、 灰黴病菌接種葵百合葉片之lpglu1 表現分析………………………..62 圖十、 水楊酸處理葵百合之lpglu1 表現分析………………………………..63 圖十一、 茉莉酸甲酯處理葵百合之lpglu1 表現分析………………………..64 圖十二、 ACC 與離層酸處理葵百合之lpglu1 表現分析…………………….65 圖十三、 傷口處理(刀傷)葵百合植株葉片之lpglu1 表現分析……………….66 圖十四、 低溫處理與高鹽處理之lpglu1 表現分析…………………………..67 圖十五、 pSK-lpg 質體圖譜…………………………………………………….68 圖十六、 E. coli DH5α (pSK-lpg)轉形株於37℃培養之粗抽蛋白質分析…..69 圖十七、 pGST-lpg 質體圖譜…………………………………………………...70 圖十八、 純化GST-lpg 融合蛋白質之分析……….…………………………...71 圖十九、 pQE-lpg 質體圖譜…………………………………………………….72 圖二十、 E. coli XL1-Blue (pQE 30-lpg)全蛋白質之西方轉漬分析…………..73 圖二十一、 不同濃度葡萄糖溶液於540 nm 吸光值之標準曲線……………75 圖二十二、 影響lpglu1 基因於葵百合植株表現之各種化學誘導因子……….76 圖二十三、 三種不同載體之重組lpglu1 DNA 序列………………………….77 圖二十四、 lpglu1 基因序列在E. coli K12 的codon usage…………………..78 | |
dc.language.iso | zh-TW | |
dc.title | 葵百合β-1,3-葡聚醣酶基因之表現分析 | zh_TW |
dc.title | Expression analysis of β-1,3-glucanase gene of Lilium cv. Star Gazer | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄭秋萍,張碧芳 | |
dc.subject.keyword | 百合灰黴病菌,系統性誘導抗病,葵百合,β-1,3-葡聚醣酶,病程相關蛋白質, | zh_TW |
dc.subject.keyword | Botrytis elliptica,B. cinerea,systemic acquired resistance,Lilium cv. Star Gazer,β-1,3-glucanase,pathogenesis-related proteins, | en |
dc.relation.page | 79 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-01-26 | |
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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