臺灣博碩士論文加值系統

油菜固醇是一植物賀爾蒙，在植物體內扮演影響生長發育的角色。近年來已有研究以核糖核酸微陣列晶片以及蛋白質體學兩種高通量實驗，檢測油菜固醇對植物整體的影響，但對於微型核醣核酸所涉及的機制仍不明瞭。我們選用哥倫比亞型的阿拉伯芥，以微型核醣核酸微陣列晶片分析油菜固醇所調控的微型核醣核酸，並發現額外添加油菜固醇後，miR395a的表現量會顯著的上升，以即時定量聚合酶連鎖反應偵測其表現，也有相同的結果。為了釐清miR395a所涉及的機制，我們整合了生物資訊以及現有的核糖核酸微陣列晶片，預測其可能的目標基因，結果顯示GUN5極有可能是miR395a的目標基因之一。GUN5是一多功能的基因，涉及植物體內代謝，包含葉綠素合成，而近年的研究更指出其可能涉及離層酸的調控路徑。離層酸是一植物賀爾蒙，參與調適植物逆境，如藉由抑制種子萌發、延長根的長度以及側根的形成。我們利用即時定量聚合酶連鎖反應，進一步分析離層酸路徑裡的基因，結果顯示GUN5下游的基因表現量下降。為了證明miR395a與GUN5是否有直接的關聯，我們利用螢光分析檢測，結果顯示在含有miR395a以及融合蛋白GUN5-GFP的組別裡，螢光蛋白的表現量下降。另外，利用miR395a promoter-GUS轉植株則顯示miR395a表現在葉脈以及根的組織中。這些結果皆顯示油菜固醇所調控的生長路徑之一，或許是藉由miR395a來抑制GUN5的表現，以阻斷離層酸路徑，並減少葉脈周邊細胞葉綠素。

Brassinosteroids (BRs) are essential plant hormones involved in growth and development. Many high-throughput researches of BRs have been provided by DNA microarray and proteomics; however, the microRNAs-involved mechanisms of BRs are still poorly understood. With the use of time-course microRNA array analysis, we identified BR-regulated microRNAs in Arabidopsis thaliana ecotype Columbia. We found that miR395a was significantly up-regulated after BR treatment, and validated the expression of miR395a and BR-regulated genes using quantitative PCR (q-PCR). In order to clarify the miR395a-involved mechanisms, we integrated bioinformatics methods and publicly available DNA microarray data to predict the potential targets of miR395a. According to these methods, GUN5 was identified as one of the possible targets to miR395a. GUN5 is a multifunctional protein involved in plant metabolism such as chlorophyll synthesis. Recent studies have shown that GUN5 might be involved in abscisic acid (ABA) pathway. ABA is a plant hormone that mediates the adaptation of plants to stress via inhibiting seed germination, root elongation and lateral root formation. We further investigated the gene expression of ABA pathway in BR-treated plants by performing q-PCR. As proposed, the downstream genes of GUN5 were down-regulated. We further used fluorescence assay to validate the relative expression ratio of miR395a and GUN5, and the result showed that the expression of GUN5-GFP fusion gene was down-regulated. Furthermore, the expression of miR395a was up-regulated in leaf veins and root tissues in BR-treated miR395a promoter-GUS plants. Our data revealed that miR395a may inhibit ABA responses by targeting GUN5, indicating that BR may affect the growth of Arabidopsis through miR395a-regulated ABA responses and causing less chlorophyll in the cell around leaf veins.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
ABSTRACT iv
CONTENTS vi
LIST OF FIGURES x
LIST OF TABLES xi
Chapter 1 Introduction 1
1.1 Arabidopsis thaliana 1
1.1.1 Global introduction 1
1.1.2 The transformation of Arabidopsis 1
1.2 Brassinosteroids 1
1.2.1 The history of BR finding 1
1.2.2 The structure of BR 2
1.2.3 BR signaling pathway 3
1.2.4 The function of BR 3
1.2.5 BR-regulated root development 4
1.2.6 BR-regulated germination 5
1.3 miRNAs 5
1.3.1 General introduction 5
1.3.2 miRNA biogenesis in plants 6
1.3.3 The function of miRNAs 7
1.4 Genomes uncoupled genes 7
1.4.1 General information 7
1.4.2 The function of GUN5 8
1.5 Motivation 9
Chapter 2 Materials and Methods 11
2.1 Plant material and growth conditions 11
2.2 Morphology analysis 11
2.2.1 Germination assay 11
2.2.2 Root growth assay 12
2.3 Comprehensive miRNA analysis 12
2.3.1 Treatment of seedlings for miRNA microarray 12
2.3.2 RNA extraction 12
2.3.3 MicroRNA microarray 13
2.3.4 The analysis of miRNA microarray 13
2.4 Real-time RT-PCR of gene and miRNA 14
2.4.1 Reverse transcription for genes 14
2.4.2 Real-time RT-PCR for genes 14
2.4.3 Reverse transcription for a specific miRNA 16
2.4.4 Real-time RT-PCR for a specific miRNA 16
2.5 Bioinformatics analysis 16
2.5.1 Predictions of novel miRNA target genes 16
2.6 Transient expression assay 17
2.6.1 Arabidopsis cell line PSB-D protoplast system 17
2.6.2 Generation the constructs for transient assay 17
2.6.3 Protoplasts preparations from PSB-D cells 18
2.6.4 Transformation of PSB-D protoplasts 18
2.6.5 Fluorescence assay for validating miR395a and GUN5 19
2.7 Generation of transgenic plants 20
2.7.1 Isolation of genomic DNA for construction 20
2.7.2 The construction of overexpressing line and promoter GUS line 20
2.7.3 Preparation the competent cells of Agrobacterium tumefaciens 21
2.7.4 Transformation of Agrobacterium 21
2.7.5 In planta transformation of Arabidopsis 22
2.7.6 The expression of miR395a promoter-GUS plants 22
Chapter 3 Results 24
3.1 Comprehensive screening of miRNA expression 24
3.2 Validation the expression of miR395a 26
3.3 Prediction of novel miRNA targets 27
3.4 Validation of the targets 27
3.5 The expression pattern of miR395a 28
Chapter 4 Discussions 29
4.1 miRNA profile 29
4.2 The function of miR395a 30
4.3 The interaction between miR395a and GUN5 31
4.4 The expression pattern of miR395a promoter-GUS 31
Chapter 5 Future Works 32
References 33
Figures 40
Tables 62
Appendix 81
Publication 83

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