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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.author | Ying-Hao Huang | en |
| dc.contributor.author | 黃英豪 | zh_TW |
| dc.date.accessioned | 2021-07-01T08:12:30Z | - |
| dc.date.available | 2021-07-01T08:12:30Z | - |
| dc.date.issued | 2002 | |
| dc.identifier.citation | 林耀輝（1994）我所瞭解的甘藷。科學農業25 : 357-360 藍逸青（1999）利用轉殖菸草分析甘藷sporamin敔動子之受傷誘導調控機制。台灣大學植物科學研究所碩士論文 An, G. (1987) Binary Ti vectors for plant transformation and promoter analysis. Methods in Enzymology 153 : 292-305 An, G., Costa, M. A. and Ha, S. B. (1990) Nopaline synthase promoter is wound inducible and auxin inducible. Plant Cell 2 : 225-233 An, G., Costa, M., Mitra, A., Ha, S-. B. and Marton, L. (1988) Organ-specific and developmental regulation of nopaline synthase promoter in transgenic tobacco plants. Plant Physiol. 88 : 547-552 An, G., Mitra, A., Choi, H. K., Costa, M. A., An, K., Thornburg, R. W. and Ryan., C. A. (1989) Functional analysis of the 3[prime]control region of the potato woumd-inducible proteinase inhibitor Ⅱ gene. Plant Cell 1 : 115-122 Baker, C. J., Orlandi, E. W., (1995) Active oxygen in plant/pathogen interactions. Annu. Rev. Phytopathol. 33 : 299-321 Barber, M. S., Bertram, R. E., Ride, J. P. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75280 | - |
| dc.description.abstract | Sporamin為甘藷塊根中含量豐富的儲藏性蛋白質，且sporamin基因的表現也可在受傷的葉片中被誘導出來。對於sporamin敔動子(簡稱PROSPOA)於轉殖菸草中的研究，也發現PROSPOA可為受傷誘導。本實驗便是針對PROSPOA上可能和受傷誘導相關的調節序列區進行分析，以鑑定PROSPOA中哪一片段含有受傷誘導的調節序列區。藉由轉殖阿拉伯芥中報告基因β-glucuronidas (GUS)的表現，來探討植株受傷或相關訊息傳導試劑處理時，PROSPOA以及不同長度的PROSPOA缺失(deletion)片段被活化的情形，期望在阿扛伯芥中能夠對於此敔動子在受傷誘導的調控機制更加瞭解，另一方面，藉由馬鈴薯再生系統的建立以及PROSPOA的轉型作用，期望能對於此敔動子是否具有組織專一性表現的特性加以探討。 先以聚合?連鎖反應(PCR)的方式進行PROSPOA缺失片段的選殖，之後將這些含有PROSPOA缺失片段嵌入含GUS報導基因的雙向載體(binary vector) pBI101中，並轉型入農桿菌(Agrobacterium tumefaciens)中，再以轉型過的農稈菌進行阿拉伯芥與馬鈴著的轉殖。藉由抗生素kanamycin篩選及PCR的確認復，獲得含有PROSPOA及不同長度的 PROSPOA缺失片段之阿拉伯芥轉殖株至少100株，選擇三週大的阿拉伯芥轉殖株以鑷子進行受傷處理，在經由GUS組織染色以及GUS螢光活性分析之後，結果顯示最短的sporamin敔動子DNA缺失片段(SPOA-del4，230 bp)的轉殖株在受傷後GUS活性有較強且顯著的表現，而且各組織的GUS活性有差異性的表現，其中以莖頂分生組織、子葉以及年輕的幼紫GUS之表現較明顯。而較長的sporamin敔動子DNA缺失片段，其表現反而有被抑制的現象。這個現象說明瞭在SPOA-del4中，似乎含有可受到受傷而誘導的序列，而在SPOA-del4序列的上游也許有一個負調控序列存在，造成較長的sporamin敔動子DNA缺失片段有被抑制的表現結果。 在以鑷子進行受傷復不同時間取樣，進行GUS的螢光活性分析，發現GUS的活性在0.5小時即有顯著的上升情況，在4?8小時達到高點，且在24小時內維持平穩的狀態，顯示sporamin敔動子(PROSPOA)在非塊根部位的表現確實與植物之受傷反應息息相關，且約在一小時即有明顯反應，以應付外來的傷害。 以各種訊息傳導相關試劑對阿拉伯芥轉殖株進行試驗，在經由GUS組織染色以及GUS螢光活性分析之後發現，全長的PROSPOA以及SPOA-dell對於茉莉酸、離層素、水楊酸以及緩衝液有誘導的作用，表現的組織主要是在莖頂分生組織以及葉子的維管束中，此外在全長的PROSPOA轉殖阿拉伯芥中，根部也有明顯的GUS活性表現。至於其他較短的sporamin敔動子DNA片段，以茉莉酸、離層素、水楊酸以及緩衝液進行處理，皆不具誘導的能力。因此,推測與這些訊息傳導試劑相關的調控序列應該位於PROSPOA的-1253?-830 bp之內。 在已建立之馬鈴薯的再生系統與轉殖系統中，誘導塊莖的形成之後，以GUS組織染色以及GUS螢光活性分析發現，在PROSPOA轉殖馬鈴著的塊莖中，有很強的GUS活性被偵測到，而在葉片組織中，GUS的活性表現並不明顯，所以sporamin放動子(PROSPOA)在馬鈴薯中是具有塊莖專一性表現的。此外，由於所選取的馬鈴薯塊莖大小有別，也發現到直徑大於0.5公分的馬鈴薯塊莖，其GUS活性表現較強，因此PROSPOA在馬鈴薯塊莖中的表現，可能跟塊莖的發育時期有所關聯。 | zh_TW |
| dc.description.abstract | Sporamin is a storage protein in the tuberous root of sweet potato, and the expression of sporamin gene can be induced in leaves by wounding-treatment. In order to identify the wounding-response element(s) in the 1.25 kb length of sporamin promoter (PROSPOA), this promoter was deleted to different lengths of DNA fragments, SPOA-dell (+l?-1130), SPOA-del2 (+l?-830), SPOA-del3 (+l?-730) and SPOA-del4 (+l?-230). After that, these DNA fragments were ligated into pBI101 vector that contained a GUS reporter gene, and all constructions were transformed into Arabidopsis mediated by Agrobacterium for promoter functional assay. Preliminary data indicated that the SPOA-del4 transgenic Arabidopsis showed the highest GUS activity after wounding-treatment. This result suggests that there could be a negative element within -230 to -830 bp (the transcription start site was indicated + l), and a putative wounding response element(s) within +1 to -230 bp of spramin promoter. Time course analysis for the wounding response of this promoter indicated that an inducible expression occurred in thirty millutes and peaked at 1to 4 hours after wounding. The GUS activities in meristem, cotyledon and young leaves are much stronger than those of other tissues. When treated with various elicitors, like JA, ABA and SA, PROSPOA::GIS and SPOA-dell::GUS do show the response. However, the expression of those shorter sporamin promoter can not be induced by those elicitors. In addition, an effort was made to prove the tissue-specific expression of spramin promoter in potato. The PROSPOA/pBI101 construction was transformed into potato, and the results suggest that the PROSPOA::GUS was expressed specifically in transgenic potato tuber. Therefore, the expression of sporamin promoter may be regulated by different developmental stages in potato. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-01T08:12:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2002 | en |
| dc.description.tableofcontents | 中文摘要…………………………………………………………………………………………………………………3 英文摘要…………………………………………………………………………………………………………………5 第一章 前言 第一節 植物的防禦機制…………………………………………………………………………………………6 第二節 植物的受傷訊息…………………………………………………………………………………………8 第三節 甘藷中的sporamin………………………………………………………………………………………12 第四節 甘藷sporamin篡因之敔動子……………………………………………………………………………14 第五節 本論文之研究目的………………………………………………………………………………………16 第二章 材料與方法 一、實驗材料………………………………………………………………………………………………………18 二、實驗方法 第一節 載體的構集…………………………………………………………………………………………19 第二節 大腸稈菌的轉型與鑑定……………………………………………………………………………24 第三節 農稈菌的轉型與鑑定………………………………………………………………………………26 第四節 阿拉伯芥之基因轉殖與轉殖株的鑑定……………………………………………………………28 第五節 轉殖阿拉伯芥中sporamin敔動子(PROSPOA)及其他缺失片段受傷誘導機制之分析 …………32 第六節 馬鈴薯之基因轉殖與鑑定…………………………………………………………………………36 第三章 結果 第一節 構築載體…………………………………………………………………………………………………45 第二節 大腸桿菌的轉型與鑑定…………………………………………………………………………………46 第三節 農稈菌的轉型與鑑定……………………………………………………………………………………48 第四節 阿拉伯芥之基因轉殖與轉殖株的鑑定…………………………………………………………………48 第五節 轉殖阿拉伯芥中sporamin敔動子(PROSPOA)及其他缺失片段受傷誘導機制之分析 ………………49 第六節 馬鈴薯之基因轉殖與鑑定………………………………………………………………………………53 第四章 討論 第一節 轉殖阿拉伯芥中PROSPOA::GUS及其他PROSPOA缺失片段的受傷誘導表現…………………………55 第二節 轉殖阿拉伯芥中PROSPOA::GUS及其他PROSPOA缺失片段對於訊息傳導物質的反應………………57 第三節 轉殖馬鈴薯中PROSPOA::GUS的表現情形………………………………………………………………60 第四節 未來展望…………………………………………………………………………………………………62 參考文獻…………………………………………………………………………………………………………………63 附圖………………………………………………………………………………………………………………………74 附錄………………………………………………………………………………………………………………………97 | |
| dc.language.iso | zh-TW | |
| dc.title | 甘藷sporamin基因啟動子上調控因數之分析研究 | zh_TW |
| dc.title | Analysis of regulatory elements of sporamin promoter from sweet potato (Ipomoea batatas Lam.) | en |
| dc.date.schoolyear | 90-2 | |
| dc.description.degree | 碩士 | |
| dc.relation.page | 100 | |
| 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 |
| Appears in Collections: | 植物科學研究所 | |
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