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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 葉開溫(Kai-Wun Yeh) | |
dc.contributor.author | Bo-Ruei Huang | en |
dc.contributor.author | 黃柏睿 | zh_TW |
dc.date.accessioned | 2021-05-20T20:02:31Z | - |
dc.date.available | 2014-08-22 | |
dc.date.available | 2021-05-20T20:02:31Z | - |
dc.date.copyright | 2011-08-22 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-16 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8847 | - |
dc.description.abstract | 南西文心蘭 (Oncidium Gower ramsey) 是亞洲的重要經濟花卉。前人研究指出,維他命C對於開花調節扮演著負調節的角色,然而,文心蘭生長在日/夜溫度30/25˚C較生長於25/20˚C環境中,明顯有較高的開花比例。因為較高的生長溫度可以有效地誘導維他命C過氧化氫酶 (OgAPX)的基因表現量與提高其活性,進而使大量的維他命C (ascorbate)被消耗掉。為了解維他命C在熱誘導開花中所扮演的角色,我們在阿拉伯芥中大量表現OgAPX。結果發現,轉殖植物(OgAPXOE)在22˚C的生長環境下,其形態上與野生型(control)並無顯著差異。然而提高其生長溫度至30˚C時,發現其較野生型有提早開花與較少的葉綠素累積。利用microarray分析受熱與維他命C共同影響的基因,發現這些基因主要種類為:氧化還原酶(oxidoreductase)、老化、防禦、訊號傳遞以及轉錄因子。進一步利用real-time PCR檢測下述之基因表現量,包括AtMYB70、At3g30720、參與circadian rhythms以及含有AP2-domain等基因。結果顯示,這些基因在較高的生長溫度中的表現情量確實較高/較低。而這些基因於vtc1 (維他命C含量僅有野生型30%的突變株)也有類似的較高表現量,而在OgPMEOX轉植株 (大量累積維他命C的轉殖株)當中卻有較低的表現量,這些證據顯示這類基因表現確實是受到內生性維他命C的含量所調控。綜合所有結果,我們推論維他命C參與在熱誘導開花過程可誘發較上游的開花相關基因之表現,上述基因皆屬於目前所有已知開花調控機制的較上游調控因子,此外,我們亦推論:在熱誘導開花中,醣類(sugar/energy)亦可能具有調節開花機制之功能。 | zh_TW |
dc.description.abstract | Oncidium Gower ramsey is an economically important cutting flower in orchid industry. Mild increase of growth temperature is effective to promote Oncidum flowering, in example of elevating day/night temperature from 25˚C/20˚C to 30˚C /25˚C. Our previous studies have revealed that higher growth temperature also induced the expression of ASCORBATE PEROXIDASE (OgAPX) and resulted in the deprivation of ascorbate (AsA) level. In order to decipher the underlying role of AsA under thermal-regulated flowering, OgAPX was overexpressed in Arabidopsis. There was no significant change on morphology between transgenic and control plants when growing under 22˚C. The OgAPXOE transgenic plants exhibited precocious flowering and reduction of chlorophyll content compared with control when growing at 30˚C condition. Microarray analyses of transgenic and control plants growing at 22˚C condition displayed differential expression patterns of genes to plants growing at 30˚C in categories in oxidoreductase, senescence, defense, signal transduction and transcription factors. AtMYB70, AP2, circadian genes and At3g30720, which displayed up/down-regulated expression pattern under elevated growth temperature condition were further validated by quantitative RT-PCR. Moreover, their expression levels were
10 associated with the endogenous AsA level and displayed up-regulation level in ascorbate-deficient mutant, vtc1, but down-regulated level in ascorbate-accumulated transgenic plants, OgPMEOX. Noteworthy, attenuated expression level of At3g30720 encoding qua-quine starch (QQS) which acting on starch biosynthesis, provides a regulatory function of AsA state on the cross-talk of thermal- and energy-regulated flowering. Taken together, our results bring about a genetic network of flowering mechanism orchestrated by the elevated growth temperature. | en |
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dc.description.tableofcontents | Table of the Content
Chinese Abstract ............................................................................................................. 8 English Abstract ............................................................................................................. 9 1. Introduction .............................................................................................................. 11 1.1 Oncidium Grower ramsey ............................................................................ 12 1.2 The Current Work on Oncidium Phase Transition .................................... 13 1.3 Regulatory Pathways of Flowering ............................................................. 16 1.3.1 Research of Flowering Time in the Pre-molecular Biology Era ................. 16 1.3.2 Regulation of Flowering by Day Length ...................................................... 17 1.3.3 Gibberellic Acid Pathway: A Hormonal-Controlled Flowering ................. 18 1.3.4 Autonomous Pathway of Flowering ............................................................. 19 1.3.5 Effect of Ambient Temperature on flowering ............................................... 20 1.3.5.1 Effect of Vernalization on Flowering ................................................... 20 1.3.5.2 Thermal-induced flowering .................................................................. 21 1.4 Ascorbate: An Emerging Field in the Regulation of Flowering Time ...... 22 1.4.1 Physiological Functions of Ascorbate ......................................................... 22 1.4.2 Ascorbate: A Negative Regulator of the Phase Transition in Planta .......... 23 1.5 The Bridge between Ascorbate and Flowering ........................................... 25 1.5.1 The Issue of Ascorbate Peroxidase .............................................................. 25 1.5.2 Hierarchy of Hydrogen Peroxide, Ascorbate and Ascorbate Peroxidase in Flowering process ......................................................................................... 26 1.6 Goals of This Thesis ..................................................................................... 27 2. Results………………………………………………………………………………. 28 2.1 Characterization of Transgenic Arabidopsis Overexpressing Oncidium Ascorbate Peroxidase .......................................................................................... 28 2.1.1 Generation of Transgenic Arabidopsis Overexpressing Oncidium Ascorbate Peroxidase ..................................................................................................... 28 2.1.2 Morphological Characterization of OgAPXOX Transgenic Plants ............ 29 2.1.3 Effects of Oncidium Ascorbate Peroxidase on Ascorbate Homeostasis and H2O2 Level under Different Environmental Condition in Arabidopsis ......... 29 2 2.1.4 Effect of Oncidium Ascorbate Peroxidase on Flowering Time under Different Environmental Condition in Arabidopsis ...................................... 31 2.2 Genetic Network of Ascorbate Peroxidase Involving in Thermal-induced Flowering Process ............................................................................................. 34 2.2.1 Decreased Expression Level of FLM in OgAPXOX Plants Suggests the Role of AsA in Regulating Thermal-induced Flowering ....................................... 34 2.2.2 Transcriptional Profiling of OgAPXOX Arabidopsis under Elevated Growth Temperature .................................................................................................. 35 2.2.3 Functional Categories of Expression Clusters in Accordance with Genotypes and Growth Temperature Condition ........................................... 45 2.2.4 Transcription Profiling Revealed That Genes Encoding MYB and AP2 TFs Expressed with Higher/ Lower in OgAPXOX Plants under Elevated Growth Temperature .................................................................................................. 46 2.2.5 Genes Involving in Circadian Rhythm and Starch Synthesis Were Significantly Altered in APXOX Plant under Elevated Growth Temperature ………………………………………………………………………..48 2.2.6 Quantification of Expression Level of Candidate Genes by Real-time PCR ................................................................................................................... 49 2.2.7 Expression Profiling of Candidate Genes in AsA Deficient or Accumulated Mutants .......................................................................................................... 51 3. Discussion…………………………………………………………………………... 52 4. Prospective ................................................................................................................ 59 5. Materials and Methods ............................................................................................ 61 5.1 Plant Materials and Growth Conditions ....................................................... 61 5.2 RNA Extraction, Microarray and qPCR ....................................................... 62 5.3 Ascorbate Peroxidase Activity Analysis ........................................................ 63 5.4 Ascorbate Measurement ................................................................................ 65 5.5 Hydrogen Peroxide Measurement ................................................................. 68 5.6 Hydrogen Peroxide Staining ......................................................................... 71 5.7 Quantitative Analysis of Protein Concentration ........................................... 72 5.8 RNA Extraction ............................................................................................. 73 5.9 RT PCR for Gene Expression (One-step RT PCR) ....................................... 77 3 5.10 Construction of Functional Plasmid for Overexpressing the Interested Genes in Arabidopsis ............................................................................................... 78 5.11 Agrobacterium Infiltration (Host: Arabidopsis thaliana Col.) ..................... 85 References ……………………………………………………………………………. 88 4 List of Tables Table 1. GO analysis of cluster 1 ................................................................................... 95 Table 2. GO analysis of cluster 2 ................................................................................... 98 Table 3. GO analysis of cluster 3 ................................................................................. 100 Table 4. GO analysis of cluster 4 ................................................................................. 101 Table 5. GO analysis of cluster 5 ................................................................................. 102 Table 6. GO analysis of cluster 6 ................................................................................. 105 Table 7. GO analysis of cluster 7 ................................................................................. 107 Table 8. GO analysis of cluster 8 ................................................................................. 110 Table 9. GO analysis of cluster 9 ................................................................................. 112 Table 10. GO analysis of cluster 10 ............................................................................. 114 Table 11. Transcriptional profiling of MYB or MYB-like transcription factors. .......... 116 Table 12. Transcriptional profiling of AP2-like transcription factors.......................... 118 Table 13. Transcriptional profiling of circadian genes. ............................................... 120 Table 14. Transcriptional profiling of QQS. ................................................................ 121 5 List of Figures Figure 1.The enzymatic activity and expression level of APX in several independent lines ........................................................................................................... 122 Figure 2. The foliar phenotype and H2O2 content in transgenic and control plants growing under long-day photoperiod and 22°C condition. ...................... 123 Figure 3. The chlorophyll content of control and transgenic plants. ........................... 124 Figure 4. The AsA level in transgenic and control plants growing under 22°C condition and long-day photoperiod. AsA content in 4 leaves of transgenic and control plants growing under 22°C condition and long-day photoperiod………………………………………………………………125 Figure 5. The H2O2 content in transgenic and control plants growing under long-day photoperiod and 22°C condition. .............................................................. 126 Figure 6. The AsA level in transgenic and control plants growing under 22°C condition and short-day photoperiod. ....................................................................... 127 Figure 7. The H2O2 content in transgenic and control plants growing under 22°C condition and short-day photoperiod. ....................................................... 128 Figure 8. H2O2 level in wild type and OgAPXOX transgenic Arabidopsis after elevating growth temperature to 30°C. .................................................................... 129 Figure 9. AsA content in transgenic plants OgAPXOX after heat treatment. .............. 130 Figure 10. The AsA redox ratio in transgenic plants OgAPXOX after elevating growth temperature to 30°C. ................................................................................. 131 Figure 11. The higher growth temperature brought about drastic decrease of AsA level in wild type and transgenic plants. ........................................................... 132 Figure 12. The number of rosette leaves at the time of flowering in OgAPXOX and control plants. ........................................................................................... 133 Figure 13. The phenotype of transgenic Arabidopsis and control plants growing under 6 short-day photoperiod and 22°C condition. .............................................. 134 Figure 14. The flowering time of transgenic Arabidopsis and control plants growing under 22°C condition and short-day photoperiod. ................................... 135 Figure 15. The phenotype of transgenic Arabidopsis and control plants growing under 30°C condition and long-day photoperiod. .............................................. 136 Figure 16. The percentage of rosette leave number when flowering in transgenic Arabidopsis and control plants growing under 30°C condition and long-day photoperiod. ............................................................................................ 137 Figure 17. OgAPXOX transgenic plants exhibited the shorter length of inflorescence stem. ........................................................................................................ 138 Figure 18. The phenotype of transgenic Arabidopsis and control plants growing under 30°C condition and short-day photoperiod. .............................................. 139 Figure 19. The percentage of rosette leave number when flowering in OgAPXOX and control plants growing under 30°C condition and short-day photoperiod. .................................................................................................................. 140 Figure 20. Relative expression levels of thermal sensitivity gene FLOWERING LOCUS M (FLM) in wild type and transgenic plants under elevated growth temperature. .............................................................................................. 141 Figure 21. Clustering analysis of 860 transcripts which displayed up- or down- regulated 2 fold levels between each samples with CK under LD-22°C at 0 day. ........................................................................................................... 143 Figure 22. Functional categories of expression clusters in accordance with genotypes and growth temperature condition. ........................................................... 144 Figure 23. Relative expression levels of AP2 transcription factors in wild type and transgenic plants under elevated growth temperature. ............................. 145 Figure 24. Relative expression levels of circadian genes in wild type and transgenic plants under elevated growth temperature. ............................................... 146 7 Figure 25. Relative expression levels of AtMYB70 and QQS in wild type and transgenic plants under elevated growth temperature. ............................................... 147 Figure 26. Relative expression levels of qua-quine starch (QQS) in three independent lines. .......................................................................................................... 148 Figure 27. Relative expression levels of AP2 transcription factors in vtc1 or OgPMEOX mutants. .................................................................................................... 149 Figure 28. Relative expression levels of circadian genes in vtc1 or OgPMEOX mutants. .................................................................................................................. 150 Figure 29. Relative expression levels of AtMYB70 and QQS in vtc1 or OgPMEOX mutants ..................................................................................................... 151 Figure 30. Proposal model of genetic network for activated APX in modulating flowering process ...................................................................................... 152 List of Appendixes Appendix 1. The oligonucleotides used for RT-PCR and qRT-PCR. ......................... 153 Appendix 2. Schematic representation of ascorbate homeostasis in pseudobulb cell of Oncidium orchid in three developmental stages. ...................................... 154 Appendix 3. Ascorbate oxidation and regeneration from monodehydroascorbate and dehydroascorbate……………………………………………………….155 Appendix 4. Abbreviation…...……………………………………………………….156 | |
dc.language.iso | en | |
dc.title | 文心蘭ascorbate peroxidase 對阿拉伯芥在中高溫生長下對於開花機制的調控功能 | zh_TW |
dc.title | Molecular function of Oncidium ascorbate peroxidase on thermal-induced flowering process in Arabidopsis thaliana | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 吳克強(Ke-Qiang Wu),陳仁治(Jen-Chih Chen),黃麗春(Li-Chun Huang),吳素幸(Shu-Hsing Wu) | |
dc.subject.keyword | 維他命C,過氧化氫酶,熱誘導開花, | zh_TW |
dc.subject.keyword | ascorbate peroxidase,thermal-induced flowering, | en |
dc.relation.page | 156 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2011-08-16 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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