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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 洪傳揚(Chwan-Yang Hong) | |
| dc.contributor.author | Nien-Chun Hsieh | en |
| dc.contributor.author | 謝念純 | zh_TW |
| dc.date.accessioned | 2021-06-16T04:12:48Z | - |
| dc.date.available | 2019-09-03 | |
| dc.date.copyright | 2014-09-03 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-20 | |
| dc.identifier.citation | 張惠如 (2005) 以病毒誘導基因沉寂策略進行菸草脯胺酸庫之代謝工程. 國立中興大學農藝學系碩士論文
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55615 | - |
| dc.description.abstract | 植物無法移動，因此必須有良好的防禦系統以避免遭受環境逆境的傷害。抗氧化系統以及脯胺酸在植物抵抗逆境均扮演重要的角色。穀胱甘肽還原酶 (Glutathione reductase, GR) 為植物重要的抗氧化酵素之一，負責將氧化態的穀胱甘肽 ( GSSG) 還原成還原態 GSH，避免細胞內氧化還原系統失衡；脯胺酸在植物中扮演滲透調節者的角色，會受到 Proline deydrogenase (ProDH) 代謝。為了了解 C4 型模式植物高粱 GR 基因以及圓葉菸草 ProDH 基因之特性，試驗中選殖並分析高粱的 3 個 GR 基因和圓葉菸草 ProDH 基因。結果顯示SbGR1 與 SbGR2 和水稻 OsGR1 及 OsGR2 胺基酸序列相似度分別達 93 % 與 95 %，SbGR3 與 OsGR3 則只有 49 %。與基因體組序列比對結果發現 SbGR3 基因中一段 Intron 未被移除，導致一個 stop codon 出現，讓此基因無法轉譯出正常的蛋白質。以大腸桿菌異源表現 SbGR，結果顯示 SbGR1 及 SbGR2 可表現出具有GR活性的蛋白質，而 SbGR3 蛋白質則不具 GR 活性；次細胞定位分析顯示 SbGR1 位於葉綠體的澱粉體，SbGR2 則位於細胞質。鹽逆境會誘導 SbGR1 及 SbGR2 表現，高溫 (45℃) 誘導 SbGR1 表現，但抑制 SbGR2 表現。這些結果顯示，水稻及高粱 GR 同源基因對高溫逆境的反應及蛋白質位置具有不同結果，此差異對抗氧化系統之影響仍有待研究。在 ProDH 基因部分，試驗中選殖出圓葉菸草的 ProDH 基因，稱為 NbProDH，基因全長為 1895 bp，ORF (Open reading frame) 為 1497bp，蛋白質分子量為 55 kDa。ProDH 表現量以葉片最高，其次是葉柄跟莖；ProDH 表現會受缺水抑制、受復水誘導，而脯胺酸會在缺水 12 小時後開始累積，試驗中亦建立大量表現 NbProDH 轉殖植株，以進行基因功能分析。 | zh_TW |
| dc.description.abstract | Plants couldn’t move, so they have developed a good defense system to avoid suffering from the damage of environmental stresse. An antioxidant defense system and proline play an important role in the resistance to stress in plants. Glutathione reductase (GR), one of the antioxidant defense systemic enzymes, mediates the reduction of oxidized glutathione (GSSG) to GSH to maintain cellular redox homeostasis. Proline is suggested to act as a compatible osmolyte and be degraded by proline dehydrogenase (ProDH). To understand thecharacteristics of GR gene in C4 model plant – sorghum and the ProDH gene in N. benthamiana, our study cloned and analized three sorghum GR genes and the N. benthamiana ProDH gene. The result showed that the amino acid sequence similarities of SbGR1 and rice OsGR1 were 93%, SbGR2 and OsGR2 were 95%, but only 49% for SbGR3 and OsGR3. Compared with the sequence of genomic DNA, we found that one fragment of intron wasn’t removed from the SbGR3 cDNA which resulted in a premature stop codon and let this gene can’t translate a normal protein. The expression of SbGR1 and SbGR2 in E. coli validated that it can be translated as a protein with GR activity. However, overexpression of SbGR3 in E. coli produced no GR activity. Subcellular localization of SbGR-GFP revealed that SbGR1 was localized to the chloroplast and SbGR2 to the cytosol. SbGR1 and SbGR2 were induced by salinity. A high temperature (45℃) induced the expression of SbGR1, but repressed the expression of SbGR2. These results show that the expression of homologous GR genes in rice and sorghum were different in response to the heat stress, and also different in the expressed protein location. Therefore, the differences of antioxidant defense systems between sorghum and rice have yet to be studied. In the other part, we isolated the proline dehydrogenase gene from Nicotiana benthamiana which called NbProDH. The full-length NbProDH gene is 1895 bp. The cloned cDNA contains open reading frames of 1497 bp and encodes protein of 499 amino acids with a molecular mass of 55 kDa. Under normal condition, the expression of NtProDH is higher in leaves than in petiole and stem. The expression of NbProDH was repressed by dehydration and induced by rehydration. And proline began to accumulate after dehydration for 12 hours. This studied also established the overexpression NbProDH transgenic plants for gene functional analysis. | en |
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| dc.description.tableofcontents | 誌謝 ……………………………………………………………......……………….…i
摘要 ……………………………………………………………………………..……ii ABSTRACT ………………………………………………………………..…..…….iii 目錄 …………………………………………………………………………..……...vi 圖目錄 .………………………………………………………………………..…....viii 表目錄 ………………………………………………………………………….……ix 附表目錄 ……………………………………………………………………….……ix 縮寫字對照表 ………………………………………………………………………..x 前言 …………………………………………………………………………………..1 第一章、圓葉菸草脯胺酸去氫酶基因選殖及特性分析 …………………………..3 一、前人研究 ………………………………………………………………………3 (一) 脯胺酸與非生物逆境之關係 ……………………………………………..3 (二) 植物脯胺酸之累積機制 …………………………………………………..4 (三) 脯胺酸去氫酶 (Proline dehydrogenase, ProDH) 功能及研究 …………..4 二、研究目的 …………………………………………………………………........6 三、材料與方法 ……………………………………………………………………7 (一) 試驗材料 ..…………………………………………………………………7 (二) 總量 RNA 萃取 …………………………………………………………..7 (三) 基因組 DNA 萃取 ………………………………………………………. 7 (四) 反轉錄聚合酶鏈鎖反應 (Reverse transcription polymerase chain reaction，RT-PCR) ..……………………………………………………………….….8 (五) 圓葉菸草 ProDH 基因選殖 …………………………………………….. 9 (六) 基因序列比對及親緣演化樹分析………………………………………... 9 (七) 質體 DNA 之萃取 ………………………………………………………. 9 (八) 載體構築 …………………………………………………………………10 (九) 大腸桿菌的轉型作用 (transformation) ………………………………….10 (十) 農桿菌轉型作用 …………………………………………………………11 (十一) 圓葉菸草轉殖 ………………………………………………………...11 (十二) 圓葉菸草乾旱及復水試驗 …………………………………………...12 (十三) 脯胺酸之測定 ………………………………………………………...12 (十四) 南方點墨分析 (Southern blot) ………………………………………..13 四、結果與討論 …………………………………………………………………..14 (一) 圓葉菸草脯胺酸 NbProDH 基因序列比對分析 ………………………14 (二) NbProDH 基因套數之確認 ……………………………………………..14 (三) NbProDH 於圓葉菸草不同組織之表現 ………………………………..15 (四) 圓葉菸草 NbProDH 受缺水及復水之影響 ………...………………….16 (五) 大量表現 NbProDH 之 N. benthamiana 轉殖株分析 ……………… ..17 五、參考文獻 ……………………………………………………………………..18 第二章、高粱榖胱甘肽還原酶基因選殖及特性分析……...……………………… 30 一、前人研究 ……………………………………………………………………..30 (一) 植物抗氧化機制 …………………………………………………………30 (二) 抗氧化防禦系統之恆定 …………………………………………………30 (三) 榖胱甘肽還原酶 (Glutathione reductase, GR) 功能及研究 …………...31 (四) Sorghum bicolor 特性及研究 …………………………………………...33 二、研究目的 ……………………………………………………………………..35 三、材料與方法 …………………………………………………………………..36 (一) 試驗材料 …………………………………………………………………36 (二) 總量 RNA 萃取 …………………………………………………………36 (三) 反轉錄聚合酶鏈鎖反應 (Reverse transcriptase polymerase chain reaction，RT-PCR) ……………………………………………………………….…...36 (四) 高粱 GR 家族基因選殖 ……………………………………………….. 36 (五) 質體 DNA 之萃取 ……………………………………………………... 36 (六) 載體構築 …………………………………………………………………36 (七) 大腸桿菌的轉型作用 (transformation) ………………………………….37 (八) 原生質體抽取、PEG 轉殖及刺細胞定位分析………………………… 37 (九) 高粱、水稻非生物性逆境處理 ………………………………………….38 (十) 蛋白質萃取與分析..……………………………………………………... 38 (十一) 西方點墨分析 Western blot …………………………………………..39 (十二) GR 酵素活性測定 …………………………………………………...40 (十三) GR 同功酵素染色分析 (Zymography assay) ……………………….40 (十四) 數據分析 ……………………………………………………………...40 四、結果 …………………………………………………………………………..41 (一) 高粱三個 GR 胺基酸序列比對分析 ………………………………….. 41 (二) 異源表現 SbGR 於大腸桿菌 …………………………………………...41 (三) SbGR1、SbGR2次細胞定位分析 ………………………………………41 (四) OsGR1、OsGR2 專一性抗體辨識高粱 SbGR1、SbGR2 蛋白 ………41 (五) 不同逆境處理之 GR 蛋白質表現分析 ……………………………….. 42 (六) 高粱 SbGR1、SbGR2 啟動子之 cis-acting element 預測分析 ……….42 五、討論 …………………………………………………………………………..43 (一) SbGR3 為不具功能性的 GR ………………………………………..…..43 (二) SbGR1 位於澱粉體表現、SbGR2 則位於細胞質表現..………………43 (三) 高粱與水稻之特性比較 …………………………………………………44 (四) SbGR 與 OsGR 於不同逆境下表現之比較 …………………………...45 (五) SbGR1、SbGR2 啟動子之特性分析…………………………………….46 六、參考文獻 ……………………………………………………………………..47 附錄 ………………………………………………………………………………..65 圖目錄 圖一、圓葉菸草 ProDH 基因及胺基酸序列。 ……………………………….....23 圖二、N. benthamiana 與其他物種 ProDH 之演化樹親緣分析圖。 ……….....24 圖三、NbProDH 與 NtProDH1、NtProDH2 胺基酸序列比對。 ………….…..25 圖四、南方點墨分析結果與圓葉菸草 ProDH 基因結構圖。 …………….........26 圖五、NbProDH 於不同組織之基因表現結果。………………………………....27 圖六、不同時間點缺水及回水處理之圓葉菸草 NbProDH 基因表現結果。…….………………………………………………..……………….…...28 圖七、N. benthamiana 轉殖系之分子鑑定。………………………………………29 圖八、高粱三個 GR 胺基酸序列比對分析。 ……………………………………56 圖九、(A) SbGR3 序列比對 (B) 高粱 GR 基因結構圖。 …………………….57 圖十、SbGR 酵素活性異源表現於大腸桿菌。 ………………………………....58 圖十一、SbGR1、SbGR2 次細胞定位。 …………………………………....……59 圖十二、利用水稻 OsGR1、OsGR2 專一性抗體辨識高粱 SbGR1、SbGR2 異源表現蛋白。 .…………………………………………………………….….. 60 圖十三、不同逆境之高粱及水稻地上部 GR1 與 GR2 蛋白質表現分析。…… 61 圖十四、PLACE 資料庫及氧化逆境相關保守調控序列預測結果。…………....62 表目錄 表一、不同物種之 ProDH 基因序列比對相同度百分比。…………………….22 表二、高粱與水稻 GR 胺基酸相同度及相似度百分比。……………..……….55 附表目錄 附表一、本論文中構築大量表現及基因表現分析所使用之引子列表……..…..63 附表二、論文所用之溶液配方…………………………………………...…….....64 | |
| dc.language.iso | zh-TW | |
| dc.title | 圓葉菸草脯胺酸去氫酶及高粱穀胱甘肽還原酶基因的選殖及特性分析 | zh_TW |
| dc.title | Cloning and characterization of tobacco (Nicotiana benthamiana) proline dehydrogenase and Sorghum (Sorghum bicolor) glutathione reductase genes | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 陳建德(Chien-Teh Chen) | |
| dc.contributor.oralexamcommittee | 古新梅(Hsin-Mei Ku),吳蕙芬(Whei-Fen Wu) | |
| dc.subject.keyword | 脯胺酸,轉基因植物,抗氧化酵素,氧化逆境,高粱,圓葉菸草, | zh_TW |
| dc.subject.keyword | proline dehydrogenase,Nicotiana benthamiana,transgenic plant,antioxidative enzymes,glutathione reductase,sorghum,oxidative stress, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-20 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| 顯示於系所單位： | 農業化學系 | |
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