臺灣蝴蝶蘭CBF基因啟動子在阿拉伯芥轉殖株中之分析

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研究生：	蔡惠雯 Tsai, Hui-Wen
論文名稱：	臺灣蝴蝶蘭CBF基因啟動子在阿拉伯芥轉殖株中之分析 Characterization of a Phalaenopsis aphrodite C-repeat binding factor (PaCBF) promoter in transgenic Arabidopsis plants
指導教授：	林彩雲 Lin, Tsai-Yun
口試委員:	曾夢蛟楊明德楊長賢
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	61
中文關鍵詞：	蝴蝶蘭、CBF
相關次數：	點閱：1 下載：0
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　　植物在生長過程中，會遭受到各種不同嚴苛的環境變化，寒冷、乾旱和高鹽是植物比較常接觸到的非生物性逆境。當暴露在這些環境之下，有些植物能適應環境而維持生長。溫度是調控植物生長的一個重要因子，低溫影響熱帶和亞熱帶植物的生長和收成。而當植物受到低溫刺激時會誘導CBF基因表現，CBF基因活化幫助植物增加對低溫的抵抗能力。
　　蘭花培育已成國際商品，臺灣蝴蝶蘭（Phalaenopsis aphrodite）是重要的產銷花卉。本論文的目的是分離並探討PaCBF1基因啟動子的活性。實驗將不同區域的啟動子片段構築於報導基因β-glucuronidase (GUS)之前，利用暫時性轉殖至柴胡細胞和穩定轉植至阿拉伯芥轉殖株的方式分析PaCBF1啟動子的強度。暫時性的GUS表現量顯示PaCBF1啟動子的強度約為花椰菜鑲嵌病毒35S啟動子的五分之一。分析阿拉伯芥轉殖株含不同區域之PaCBF1啟動子的強度約為CaMV35S的三分之一。除了含−515到＋74區域的轉植株呈現失去啟動子活性。基因槍轉殖的結果顯示PaCBF1啟動子在臺灣蝴蝶蘭中不會有GUS活性的表現。這些實驗結果顯示PaCBF1啟動子的−701到−515區域可能含有會被阿拉伯芥中的轉錄因子所調控的重要元素序列。分析阿拉伯芥轉殖株在非生物逆境之下的表現顯示低溫能夠活化PaCBF1啟動子的表現。

Table of contents
摘要    i
Abstract    ii
謝誌    iv
Table of contents    v
List of Tables    ix
List of Figures     x
Abbreviations    xii
Introduction    1
Materials and Methods    7
1. Plant growth condition    7
●Orchid (Phalaenopsis aphrodite) plant culture    7
●Bupleurum kaoi cell suspension culture    7
●Transgenic Arabidopsis thaliana plants    7
2. Stress treatments    8
3. Genomic DNA extraction    8
●Orchid genomic DNA extraction using the CATB method    8
●Genomic DNA extraction from transgenic Arabidopsis thaliana plants    10
4. Total RNA extraction    10
5. Cloning of PaCBF1 promoter and identification of its gene transcription start site    11
●Obtaining the 5'-flanking region of PaCBF1    11
●Identification of the transcription start site    12
●DNA ligation    12
● Competent cell preparation    12
●Bacterial transformation    13
● Mini-preparation of plasmid DNA    13
● Double-stranded DNA sequencing    13
●Construction of the PaCBF1 promoter and its 5'-deletion fragments and fused to the GUS reporter gene    14
6. Large- scale plasmid DNA preparation    14
7. PEG-mediated protoplast transformation    15
●Preparation of B. kaoi protoplasts    15
●PEG transformation of protoplasts    16
●Transient transactivation assay in B. kaoi protoplast    17
8. Quantification of RLUC (p35S::RLUC) and GUS activity in transformed protoplasts    17
●Renilla Luciferase® assay    17
●Measuring β-glucuronidase (GUS) activity    17
9. Histochemical staining and fluorometric quantification of GUS 
activity in transgenic plants    18
●Histochemical staining of GUS acitivity    18
●Fluorometric analysis of GUS activity    19
10. Semi-quantitative RT-PCR    19
11. Biolistic transfection    20
Results    22
1. Isolation of the Phalaenopsis aphrodite PaCBF1 promoter    22
2. PaICE1 transactivated the PaCBF1 promoter under cold stress    23
3. PaICE1 bound and transactivation the 4×MYC element in the PaCBF1 promoter    24
4. Quantitative analysis of the PaCBF1 promoter in B. kaoi cells    24
5. Expression analyses of Arabidopsis plants carrying the PaCBF1 promoter and GUS fusion constructs    25
6. Deletion analysis of the PaCBF1 promoter in transgenic plants    27
7. The PaCBF1 promoter did not activate GUS gene in homologous transformants    29
8. Activity of the PaCBF1 promoter in transgenic Arabidopsis is affected by abiotic stresses    29
9. Low temperature (4°C) activated the PaCBF1 promoter in transgenic Arabidopsis plants    30
Discussion    32
References    36
Tables    43
Figures    45
                                

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