臺灣博碩士論文加值系統

古氏-蛋白質是由一個質量為70千道頓與一個質量為80千道頓的蛋白質所組成的異型雙次體。這種類似蛋白質被發現在從酵母菌到人類之真核生物中。多功能古氏-蛋白質參與了許多細胞代謝功能，像是轉錄調控，染色體末端結構維持以及細胞週期調控。此外，身為一個開口結合蛋白的古氏-蛋白質作用在去氧核甘酸複製的初期。植物的古氏-蛋白質基因最近在阿拉伯芥菜中被發現。當有去氧核甘酸破壞劑的刺激下，阿拉伯芥古氏-蛋白質扮演了在非同源雙股末端結合的去氧核甘酸雙股斷裂修復的角色以及如果失去了古氏-蛋白質則會造成染色體末端長度控制的失調。阿拉伯芥80千道頓古氏-蛋白質次體在T-DNA嵌合的參與也被證明出。另外，類似華納癓候的阿拉伯芥核酸外切酵素 (AtWEX) 與阿拉伯芥古氏-蛋白質之間的關係被確認了。然而，直到目前有關它在植物上的功能及調控之研究仍然是有限的。
在我們研究的第一部份，古氏-蛋白質基因從綠豆下胚軸中被篩選出來。電腦資料顯示出許多有關植物古氏-蛋白質相關訊息，像是不同物種之古氏-蛋白質的演化關係、它的預測結構、與它結合的蛋白以及在不同發育階段下阿拉伯芥古氏-蛋白質基因的表現等等。
第二部份，我們繼續探討植物古氏-蛋白質基因如何受植物荷爾蒙-生長素的調控。兩個綠豆古氏-蛋白質次體基因在綠豆中的每個組織都有表現，但以下胚軸及葉子中最多。綠豆古氏-蛋白質基因表現會受生長素依時間及劑量的依賴模式下所刺激。這種刺激會被生長素流入載體抑制劑-萘基鄰氨甲酰苯甲酸和三碘苯甲酸所減弱，表示外加的生長激素的作用。更進一步，利用專一性的生長素訊號抑制劑發現綠豆古氏-蛋白質基因在二氯苯氧機乙酸刺激下會被細胞內鈣離子敖合劑，攜鈣素拮抗劑和鈣調素抑制劑所抑制，表示攜鈣素參與在這個調節路徑中。另外，外加性的吲哚乙酸和α-萘乙酸則是透過細胞絲裂原活化蛋白激酶/细胞外信號調節激酶路徑來調節綠豆古氏-蛋白質基因的表現。綜合來說，二氯苯氧機乙酸和吲哚乙酸(或α-萘乙酸)是透過不同的路徑來調節豆古氏-蛋白質基因的表現。
第三部份，我們又繼續探討三週大阿拉伯芥菜之古氏-蛋白質基因如何受另一種植物荷爾蒙-離層酸造成之緩慢生長下的調控。首先，生物資訊分析指出有離層酸反應片段及AREB6 和ATHB5轉錄因子的結合位置在阿拉伯芥菜古氏-蛋白質基因的啟動子序列中。在轉殖的阿拉伯芥古氏-蛋白質基因啟動子之活性分析中指出離層酸使古氏-蛋白質基因啟動子的活性減少。阿拉伯芥古氏-蛋白質基因表現會受離層酸依時間及劑量的依賴模式下所抑制。然而，加了離層酸生合成抑制劑-fluride 和tungate，卻不能破壞離層酸對阿拉伯芥古氏-蛋白質基因的抑制。此外，利用訊號抑制劑處理及離層酸反應突變株分析可知，阿拉伯芥古氏-蛋白質基因受離層酸的調控是藉由細胞外鈣離子、磷脂酶D alpha、p38類型細胞絲裂原活化蛋白激酶、細胞絲裂原活化蛋白激酶6和離層酸轉錄因子-ABI3和ABI5的路徑。最後，離層酸和其拮抗性荷爾蒙(生長素和吉貝素)之間對阿拉伯芥古氏-蛋白質基因的調控路徑是沒有交互作用的。
最後，我們著重在探討阿拉伯芥菜之古氏-蛋白質基因如何受環境逆境-熱休克的調控。首先，生物資訊分析指出有熱休克反應片段及熱休克轉錄因子的結合位置在阿拉伯芥菜古氏-蛋白質基因的啟動子序列中。利用即時反轉錄聚合酶連鎖反應和轉殖之阿拉伯芥古氏-蛋白質基因啟動子活性分析，三週大阿拉伯芥菜古氏-蛋白質基因表現會受熱逆境依時間的依賴模式下所抑制。另一方面，利用離層酸生合成突變株-aba3和逆相高效液相層析法分析指出阿拉伯芥古氏-蛋白質基因抑制的減弱以及離層酸生合成的增加，顯示出阿拉伯芥古氏-蛋白質基因受熱逆境的調控是藉由離層酸生合成的路徑。乙烯訊號傳遞、去氧核甘酸修復路徑和脂肪酸生合成也參與了阿拉伯芥古氏-蛋白質基因在熱逆境下的調控。更進一步，我們的結果指出熱逆境抑制較年輕植株中阿拉伯芥古氏-蛋白質基因的表現但卻促進較老植株中阿拉伯芥古氏-蛋白質基因的表現。
總結來說，生物資訊工具及網路資料庫給了我們更多與植物阿拉伯芥古氏-蛋白質有關的知識並且實驗結果也提出了植物阿拉伯芥古氏-蛋白質基因在受不同生長素，離層酸和熱休克調節之分子層次上的證據。

Ku is a heterodimer consisting of two related subunits, Ku70 and Ku80. Orthologues of both subunits have been found in many eukaryotes from yeast to man. This multifunctional protein is involved in many cellular metabolic processes, such as transcriptional regulation, telomeric maintenance, and cell cycle regulation. Moreover, Ku, being an origin binding-protein, acts at the initiation step of DNA replication. Plant Ku genes were recently identified in Arabidopsis thaliana (AtKu). AtKu has role in the repair of DNA double-strand breaks by non-homologous end joining in response to DNA damaging agents and lack of AtKu results in a deregulation of telomere length control. Involvement of AtKu80 in T-DNA integration was also verified. Moreover, an interaction between the Werner syndrome-like exonuclease AtWEX and the AtKu heterodimer was identified. However, studies about its function and regulation in plant are still limited until now.
Firstly, the cDNAs encoding Ku70 (VrKu70) and Ku80 (VrKu80) were isolated from mung bean (Vigna radiata L.) hypocotyls. Computational analysis showed a lot of information about plant Ku, such as phylogenetic relations of Ku proteins among different organisms, predicted its structure. The Ku-interacting proteins and Ku expression under different environmental conditions were studied.
Secondly, we investigated how plant Ku is regulated by plant hormone, auxin. Both VrKu genes were expressed widely among different tissues of mung bean with the highest levels in hypocotyls and leaves. The VrKu gene expression was stimulated by exogenous auxins in a concentration- and time-dependent manner. The stimulation could be abolished by auxin transport inhibitors, N-(1-naphthyl) phthalamic acid and 2,3,5-triiodobenzoic acid, implicating that exogenous auxins triggered the effects. Further analysis using specific inhibitors of auxin signaling showed that the stimulation of VrKu expression by 2,4-dichlorophenoxyacetic acid (2,4-D) was suppressed by intracellular Ca2+ chelators, calmodulin antagonists, and calcium/calmodulin dependent protein kinase inhibitors, suggesting the involvement of calmodulin in the signaling pathway. On the other hand, exogenous indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) stimulated VrKu expression through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Altogether, it is thus proposed that 2,4-D and IAA (or NAA) regulate the expression of VrKu through two distinct pathways.
Thirdly, we continuously studied how AtKu is regulated during abscisic acid (ABA) induced slow growth in three-week-old seedlings. Bioinformatic analysis firstly predicted the existence of several ABA responsive elements and binding sites of transcription factor, AREB6 and ATHB5, on AtKu promoters. AtKu promoter-β-glucuronidase (GUS) analysis in transgenic Arabidopsis showed reduced activity of AtKu promoter upon ABA application. AtKu gene repression by ABA treatment is in a time- and concentration-dependent manner using GUS assay and real time quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. However, addition of ABA biosynthesis inhibitors, fluride and tungate, could not abolish the AtKu suppression. Moreover, AtKu repression in response to ABA was mediated through the pathway of extracellular Ca2+, phospholipase D alpha, p38-type mitogen-activated protein kinase (MAPK), MAPK6 and ABA transcription factors, ABI3 and ABI5 by analysis of inhibitor treatments and ABA responsive mutants. Finally, no cross-talk was found for modulating AtKu gene expression between ABA and antagonist hormones (auxins and gibberellic acid).
Finally, we focused on how AtKu is regulated by environmental stress-heat shock. Bioinformatic analysis found several heat shock responsive elements and heat shock transcription factor binding sites on AtKu promoters. The expression of AtKu is down-regulated by heat stress in a time course with real time RT-PCR and AtKu promoter-GUS (β-Glucuronidase) analysis using 3-week-old young seedlings. On the other hand, the high-temperature repression of AtKu is mediated through ABA biosynthesis, as shown by reversed repression of the AtKu in ABA-biosynthesis mutant, aba3 and increased ABA level analyzed by reverse high performance liquid chromatography. The involvement of ethylene signaling, DNA repair pathway and fatty acid synthesis in AtKu regulation by heat were also shown. Furthermore, our results showed heat regulated tissue-specific AtKu repression at different developmental stages.
Taken together, bioinformatics tools and online databases gave us extended knowledge about functions of plant Ku protein. In addition, the experimental results provided molecular evidence for plant Ku gene regulation by plant hormones and stress.

ACKNOWLEDGEMENTS ---------------------------------------I
ABSTRACT (IN CHINESE) --------------------------------III
ABSTRACT (IN ENGLISH) ---------------------------------VI
ABBREVIATIONS -----------------------------------------IX
LIST OF FIGURES --------------------------------------XIV
LIST OF TABLES -------------------------------------XVIII

1.INTRODUCTION ----------------------------------------1

2. MATERIALS AND METHODS ------------------------------6
2.1. Molecular Cloning and Sequence Analysis of
VrKu cDNA ---------------------------------------6
2.2. Expression and Purification of Plant Ku --------7
2.3. Bioinformaitc Tools and Online Database --------8
2.4. Antibody Preparation ---------------------------9
2.5. Chemicals --------------------------------------9
2.6. Plant Materials and Chemical/Stress
Treatments ------------------------------------10
2.7. GUS Assay -------------------------------------11
2.8. RNA Isolation ---------------------------------12
2.9. Reverse Transcription Polymerase Chain
Reaction and Quantitative Real-time PCR -------12
2.10. Protein Extraction and Immunoblotting
Analysis -------------------------------------13
2.11. Electrophoretic Mobility-shift Assay ---------14
2.12. ABA Level Analysis ---------------------------15

3. RESULTS -------------------------------------------16
3.1. Cloning and Bioinformatic Characterizations
of Plant Ku------------------------------------16
3.1.1. Molecular Cloning of the Putative VrKu70
and VrKu80 cDNAs ---------------------------16
3.1.2. Bioinformatic Characterizations
of Plant Ku---------------------------------17
3.1.2.1. Sequence Comparison ---------------------17
3.1.2.2. Sequence Analysis -----------------------18
3.1.2.3. Transcriptome Analysis ------------------19
3.2. Differential Regulation of Ku Gene Expression
in Etiolated Mung Bean Hypocotyls by Auxins ---20
3.2.1. Expression of VrKu in Response to Auxins----20
3.2.2. Differential Regulation of VrKu Expression
in Response to Auxins ----------------------23
3.3. Signaling Pathways Mediating the Suppression
of Arabidopsis thaliana Ku Gene Expression by
Abscisic Acid ----------------------------------25
3.3.1. ABA-responsive Elements on AtKu Promoters
and the Modulation of Promoter Activity
by ABA -------------------------------------26
3.3.2. Down-regulation of AtKu Gene Expression by
ABA ----------------------------------------27
3.3.3. Signaling Pathways Mediating AtKu Gene
Suppression by ABA---------------------------29
3.3.4. Modulation of Plant Hormones for AtKu
Expression ----------------------------------31
3.4. Regulation of Arabidopsis thaliana Ku Genes at
Different Developmental Stages under
Heat Stress ------------------------------------32
3.4.1. Heat Shock-Responsive Elements on
AtKu Promoter and AtKu Promoter-GUS Fusion
Inactivation by Heat ------------------------32
3.4.2. Down-regulated of AtKu Expression by Heat---33
3.4.3. Signaling Pathways for AtKu Repression
by Heat Stress------------------------------34
3.4.4. AtKu Expression at Different Developmental
Stages in Response to Heat ------------------35

4. DISCUSSIONS ---------------------------------------36
4.1. Cloning and Bioinformatic Characterizations of
Plant Ku ---------------------------------------36
4.2. Differential Regulation of Ku Gene Expression
in Etiolated Mung Bean Hypocotyls by Auxins----37
4.3. Signaling Pathways Mediating the Suppression
of Arabidopsisthaliana Ku Gene Expression by
Abscisic Acid ---------------------------------42
4.4. Regulation of Arabidopsis thaliana Ku Genes at
Different Developmental Stages under
Heat Stress ------------------------------------45

5. CONCLUSION ----------------------------------------49

6. REFERENCES ----------------------------------------50

7. FIGURE LEGENDS ------------------------------------65
7.1. Cloning and Bioinformatic Characterizations of
Plant Ku --------------------------------------65
7.2. Differential Regulation of Ku Gene Expression
in Etiolated Mung Bean Hypocotyls by Auxins ---70
7.3. Signaling Pathways Mediating the Suppression
of Arabidopsisthaliana Ku Gene Expression
by Abscisic Acid ------------------------------80
7.4. Regulation of Arabidopsis thaliana Ku Genes
at Different Developmental Stages under
Heat Stress------------------------------------89
7.5. Conclusion ------------------------------------95

8. FIGURES -------------------------------------------97
8.1. Cloning and Bioinformatic Characterizations
of Plant Ku -----------------------------------97
8.2. Differential Regulation of Ku Gene Expression
in Etiolated Mung Bean Hypocotyls by Auxins ---124
8.3. Signaling Pathways Mediating the Suppression
of Arabidopsis thaliana Ku Gene Expression by
Abscisic Acid ---------------------------------141
8.4. Regulation of Arabidopsis thaliana Ku Genes at
Different Developmental Stages under
Heat Stress------------------------------------155
8.5. Conclusion ------------------------------------169

9. TABLES---------------------------------------------170
9.1. Cloning and Bioinformatic Characterizations
of Plant Ku -----------------------------------170
9.2. Signaling Pathways Mediating the Suppression
of Arabidopsis thaliana Ku Gene Expression by
Abscisic Acid ---------------------------------180
9.3. Regulation of Arabidopsis thaliana Ku Genes
at Different Developmental Stages under
Heat Stress------------------------------------188

10. APPENDIXES ---------------------------------------182
10.1. Plasmid Constructs ---------------------------182
10.1.1. Constructs for Sequencing Full Length
of VrKu Gene.------------------------------182
10.1.2. Constructs for Expression of His6 Fusion
Proteins. ---------------------------------183
10.1.3. Constructs for Expression of GST Fusion
Proteins. ---------------------------------184
10.2. Chemicals ------------------------------------185
10.3. Primer Sets ----------------------------------186
10.3.1. Primers for Ku Fusion Protein Expression.-186
10.3.2. Primers for RT-PCR. ----------------------187
10.3.3. Primers for Real-time RT-PCR.-------------188
10.4. Arabidopsis Mutants --------------------------189

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