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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 洪傳揚(Chwan-Yang Hong) | |
| dc.contributor.author | Kuan-Yu Chen | en |
| dc.contributor.author | 陳寬宇 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:04:32Z | - |
| dc.date.copyright | 2014-09-03 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-20 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18424 | - |
| dc.description.abstract | 孔雀草(Tagetes patula)是一種常見的景觀花卉，已被證實為一種鎘的累積植物。我們過去的研究發現鎘會誘導孔雀草抗壞血酸過氧化酶(Ascorbate peroxidase, APX)活性增加APX是重要的抗氧化酵素，可分解H2O2，為了解APX對孔雀草鎘耐受性扮演的角色，本試驗進行孔雀草APX基因的選殖與功能分析。試驗中利用RACE-PCR方法，從孔雀草中選殖出一個由251個胺基酸組成的APX全長cDNA，命名為TpAPX1。該基因具有8個外顯子與7個內顯子，演化樹分析顯示其與草莓的APX親緣性最接近。TpAPX1在花有較高表現，老葉最少，受CdCl2 誘導，但受到銅與三價砷的抑制。功能分析部分，在大腸桿菌大量表現TpAPX1可表達出具有APX活性蛋白。次細胞定位分析顯示TpAPX1表現在細胞質。大量表現TpAPX1在阿拉伯芥中，可使轉殖阿拉伯芥在100μM CdCl2 與0.1 μM Methyl Viologen中，具有比一般植株(WT)較高的逆境抗性，顯示TpAPX1具有幫助植物抵抗氧化逆境的功能。 | zh_TW |
| dc.description.abstract | Tagetes patula, a common landscape plant, has been identified to be a cadmium (Cd)-accumulator. According to our previous study, the activity of ascorbate peroxi-dase (APX) in T. patula can be induced by CdCl2 treatment. APX that can detoxify H2O2 using ascorbate as a substrate plays a key role in protect on plants from oxidative stress. To evaluate the physiological role of APX in T. patula under CdCl2 treatment, rapid amplification of cDNA ends (RACE) method was used to clone the APX gene in T. patula. The full-length cDNA of TpAPX1 contains a 753 bp open reading frame which encodes 251 amino acids with an estimated molecular weight of 25 kDa. The genomic structure of TpAPX1 has 8 exons and 7 introns. Phylogenetic analysis revealed TpAPX1 has high similarity with Fragaria x ananassa APX. TpAPX1 was expressed in all tissues of TpAPX1 , and the highest and lowest mRNA levels were in flowers and roots, respectively. TpAPX1 mRNA level was induced by CdCl2 but
repressed by CuSO4 and As2O3. The expression of TpAPX1 in E. coli validated that it can be translated as a protein with APX activity. PEG-mediated transient expression with a TpAPX1-GFP fusion construct in Arabidopsis protoplast shwowed that TpAPX1 localizes in cytosol. Ectopic expression of TpAPX1 in Arabidopsis increased Methyl Viologen and Cd2+ stress tolerance. In conclusion, cytosolic TpAPX1 plays an important role of oxidate stress tolerance in plant. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:04:32Z (GMT). No. of bitstreams: 1 ntu-103-R01623021-1.pdf: 1868967 bytes, checksum: 2a43c6cebbb9a8bd0830aaf59e87e3ec (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 目錄 2
摘要 5 Abstract 6 縮寫字對照表 7 壹、前言 8 1.植物與氧化逆境 8 1.1.氧化逆境的生成與影響 8 1.2.植物的抗氧化系統 8 2.鎘 12 2.1.鎘毒害 12 2.2超累積植物 14 2.3鎘誘導氧化逆境 15 2.4 孔雀草 Tagetes putala 16 貳、研究目的 17 參、材料與方法 18 1.試驗材料 18 2.未知基因選殖 18 3.質粒的構築 22 4.質粒製備 23 5.大腸桿菌(E.coli)質粒DNA純化 24 6.阿拉伯芥轉殖 25 7.APX逆境誘導分析 28 8.次細胞定位分析 31 肆、結果 32 1.孔雀的APX基因選殖 32 2.APX基因表現分析 33 3.TpAPX1功能分析 33 伍、討論 35 陸、結論 38 未來展望 39 捌、引用文獻 40 玖、圖 46 圖1、利用RACE選殖出孔雀草的APX基因(A)不同物種APX保守區位，黑色實線為引子設計區位；(B)RACE選殖之電泳結果。 46 圖2、TpAPX1 cDNA全長序列，” * ”表示起始密碼子，” - “表示為終止密碼子。圈圈表示功能性胺基酸，框框表示功能性區位。(APLMLRLAWHSA)為APX的活性位點序列 (peroxidases active site motif)；155-165 (DIVTLSGGHTL)為過氧化氫與血紅素結合位點序列 (peroxidases heme ligand motif) 47 圖3、APX胺基酸序列比對結果。由6個不同物種cytosolic isform APX 與孔雀草APX胺基酸序列進行比對，三角形代表APX的保守胺基酸。(物種請參照附錄3) 48 圖4、TpAPX1基因序列及結構。( A)基因體組序列，小寫字母內顯子序列。B. TpAPX1基因cDNA與染色體組DNA的結構，黑色為外顯子，白色為內顯子。 49 圖5、利用RACE選殖出孔雀草的UBQ10基因(A)不同物種UBQ保守區，黑色實線為引子設計區位；(B)RACE選殖的電泳結果，框框內的條帶為目標基因之條帶。 50 圖6、TpUBQ10 cDNA全長序列，” * ”表示起始密碼子，” - “表示為終止密碼子。 51 圖7、TpAPX1之演化樹親緣分析。利用neighbor joining 方法，bootstraps value 為1000 繪製出TpAPX1 與其他物種同源基因的演化關係圖。(物種完整學名與登入碼accession number 如附錄3) 52 圖8、孔雀草在自然狀況下，水耕栽培42天，取剛抽出的新葉 (New leaf)、子葉與第一對葉 (Old leaf)、未開花之花苞 (Flower)、成熟花內的種子 (Seed)與根尖部位 (Root)等，並以RT-PCR分系各同組織TpAPX1的表現分析。 53 圖9、以14天大的孔雀草小苗分別處理鎘、銅、鋅、鉛、鉻與砷，由低到高各五個不統的濃度，處理2小時後，進行TpAPX1 RT-PCR分析。(A)地上部(B)地下部。 54 圖10、以14天大的孔雀草小苗各別進行鎘、鹽與低溫處理，並分析不同時間點下TpAPX1 表現量(A) 10 μM Cd處理下，不同時間點TpAPX1表現分析；(B)150 mM 氯化鈉處理之不同時間點TpAPX1表現分析；(C) 4℃處理之不同時間點TpAPX1表現分析。 55 圖11、TpAPX1在大腸桿菌系統大量表現分析結果。(A)APX活性分析；(B)大量表現APX蛋白質染色結果；(C)Zymogram APX 活性分析， 56 圖12、TpAPX1次細胞定位分析。圖為阿拉伯芥的原生質體，綠色為螢光蛋白發出的螢光，紅色為葉綠體的自發螢光。上圖(GFP)為只有GFP構築的表現結果，下圖為(TpAPX-GFP)為TpAPX1 於C端接上GFP構築的表現結果。 57 圖13、OE TpAPX1轉殖植物分析。挑選1-1、2-3、3-3轉殖系的T3代種子，種種植三週挑選葉片進行分析(A)西方墨點分析；(B)APX Zymogram分析。 58 圖14、大量表現TpAPX1轉殖植物鎘耐受性分析。挑選1-1、2-3、3-3轉殖系的T3代種子，種植於含0、50 μM、100 μM 的1/2MS 培養基中。 59 圖15、大量表現TpAPX1轉殖植物MV耐受性分析。挑選1-1、2-3、3-3轉殖系的T3代種子，種植於含0、0.05 μM、0.5 μM 的1/2MS 培養基中。 60 圖16、轉殖用質粒構築設計。A、大量表現目標基因於大腸桿菌系統的構築。B、將報導基因GFP接皆在目標基因C端，為次細胞定位分析的構築。C、大量表現目標基因於阿拉伯芥的構築。 61 拾、附錄 62 附錄1.培養基 62 附錄2. 實驗藥品 64 附錄3 各物種縮寫與登入碼 70 附錄.4引子列表 71 | |
| dc.language.iso | zh-TW | |
| dc.title | 孔雀草抗壞血酸過氧化酶基因之選殖與功能分析 | zh_TW |
| dc.title | Molecular Cloning and Functional Characterization of an Ascorbate Peroxidase Gene from Tagetes Patula L. | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張孟基(Men-Chi Chang),蔡育彰(Yu-Chang Tsai),陸重安(Chang-An Lu),黃文理(Wen-Li Huang) | |
| dc.subject.keyword | 孔雀草,鎘,抗壞血酸過氧化氫?,cDNA 末端快速放大法, | zh_TW |
| dc.subject.keyword | Tagetes patula,cadmium,Ascorbate peroxidase,Rapid amplification of cDNA ends (RACE), | en |
| dc.relation.page | 71 | |
| dc.rights.note | 未授權 | |
| 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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