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  1. NTU Theses and Dissertations Repository
  2. 生命科學院
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請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75335
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dc.contributor.authorHsiu-Chin Chenen
dc.contributor.author陳秀卿zh_TW
dc.date.accessioned2021-07-01T08:12:43Z-
dc.date.available2021-07-01T08:12:43Z-
dc.date.issued2001
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75335-
dc.description.abstract超氧歧化?(Superoxide dismutase; SOD)是一群含金屬的酵素,依其所含金屬不同可分為三類:FeSOD、MnSOD及CuZnSOD,其功能是清除超氧自由基。阿拉伯芥之CuZnSOD至少有三個同功?,各分佈於細胞質、葉綠體及過氧化體。本論文利用基因轉殖技術將阿拉伯芥細胞質型CuZnSOD (CSD1)轉殖於阿拉伯芥植株,得到7株預期CuZnSOD活性提高之T1轉殖株(ACZ1系列),經蛋白質電泳分析SOD活性,這些轉殖株之CuZnSOD活性增加量並未如預期大量提高。另外CuZnSOD活性下降之T1轉殖株32株,其中8株是利用反轉譯股方式得到(ACZ1a系列)、其餘24株則是利用RNAi方式產生(ACZ1i系列),其CuZnSOD活性下降由0-78%不等。觀察野生型、轉殖株及CZ5(實驗室已有之轉殖株,含水稻細胞質型CuZnSOD, SOD總活性可提高約2.7倍),發現CuZnSOD活性下降之轉殖株(ACZ1i,ACZ1a)的果莢較短、種子數目較少。另外利用CuZnSOD活性不同之轉植株(ACZ1i系列、CZ5及野生型)進行凍害逆境處理(-3℃),測量其Fv/Fm可反應光合作用效率,做為凍害耐受性指標,處理前與處理後Fv/Fm變化值顯示提高阿拉伯芥葉片CuZnSOD活性可增加阿拉伯芥葉片對凍害耐受性。zh_TW
dc.description.abstractSuperoxide dismutase (SOD) is a metalloprotein and function as free radical scavenger. Depend on the bounded metal, SOD can be divided into FeSOD, CuZnSOD and MnSOD. There are three CuZnSOD isozymes in Arabidopsis thaliana and distribute in various location of cells, including cytosol, chloroplast and peroxisome. In order to determine the role of CuZnSOD in plant reproductive stage and freezing tolerance, we generated 7 overexpression (ACZ1) and 32 CuZnSOD reduction transformants by the Agrobacterium-mediated transformation method. Within these 32 transgenic plants, 8 are generated by antisense method (ACZ1a) and the other 24 are by RNAi method (ACZli). Comparing with the wild type, cytosolic CuZnSOD activities in ACZ1 transformants do not significantly increase. However, in ACZ1a and ACZli decrease 0-78%. The length of siliques and the seed number per siliques of ACZ1a and ACZli are shorter and fewer than these of wild type. To further observe the effect of the CuZnSOD activity in freezing tolerance, freezing treatment (-3℃) is imposed to wild type, ACZli and CZ5 plants. Photosynthesis efficiency showed by Fv/Fm value of each plant is taken as the freezing tolerance indicator. The result suggests that the higher CuZnSOD activity transformants have higher freezing tolerance.en
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dc.description.tableofcontents致謝……………………………………………………Ⅰ
目錄……………………………………………………Ⅱ
縮寫表……………………………………………………Ⅳ
中文摘要……………………………………………………Ⅵ
英文摘要……………………………………………………Ⅶ
第一章 前言……………………………………………………1
第二章 材料與方法……………………………………………………7
一、質體之構築與製備……………………………………………………7
1.質體之抽取……………………………………………………7
2.PCR反應……………………………………………………8
3.pPZPACZ1i質體構築……………………………………………………9
4.接合反應……………………………………………………10
5.大腸桿菌competent cell製備……………………………………………………11
6.大腸桿菌之轉殖……………………………………………………11
7.農桿菌competent cell(C58)製備……………………………………………………12
9.農桿菌之轉殖……………………………………………………13
二、阿拉伯芥栽種與轉殖株之篩選
1.阿拉伯芥種植……………………………………………………13
2.阿拉伯芥轉殖-----浸泡法轉殖……………………………………………………14
3.轉殖株篩選……………………………………………………15
三、DNA抽取與南方(Southern)分析法
1.植株DNA抽取……………………………………………………16
2.電泳與轉印……………………………………………………17
3.Dig標定探針製備……………………………………………………18
4.訊息偵測……………………………………………………19
四、蛋白質製備……………………………………………………20
1.蛋白質萃取……………………………………………………20
2.蛋白質定量……………………………………………………21
五、酵素活性分析
1.聚丙烯硫胺膠體電泳……………………………………………………21
2.超氧歧化每活性染色……………………………………………………22
3.Catalase活性染色……………………………………………………23
4.Ascorbate peroxidase活性染色……………………………………………………23
六、凍害處理……………………………………………………24
第三章 結果
一、植株篩選與鑑定……………………………………………………26
二、SOD活性分析……………………………………………………35
三、T1轉殖株與野生型植株生長與發育觀察……………………………………………………36
四、凍害處理……………………………………………………49
第四章 討論
一、ACZ1i、ACZ1a與ACZ1轉殖株CuZnSOD活性表現……………………………………………………51
二、CuZnSOD活性對植株生殖期影響……………………………………………………52
三、CuZnSOD活性與凍害抗性具正相關性……………………………………………………53
第五章 未來展望與研究方向……………………………………………………55
第六章 參考文獻……………………………………………………56
dc.language.isozh-TW
dc.title阿拉伯芥CuZnSOD生理功能之研究zh_TW
dc.titleStudy of CuZnSOD Physiological Function in Arabidopsis thalianaen
dc.date.schoolyear90-2
dc.description.degree碩士
dc.relation.page64
dc.rights.note未授權
dc.contributor.author-dept生命科學院zh_TW
dc.contributor.author-dept植物科學研究所zh_TW
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