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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭石通 | zh_TW |
| dc.contributor.advisor | Shih-Tong Jeng | en |
| dc.contributor.author | 劉上源 | zh_TW |
| dc.contributor.author | Shang-Yuan Liu | en |
| dc.date.accessioned | 2024-02-22T16:44:55Z | - |
| dc.date.available | 2024-02-23 | - |
| dc.date.copyright | 2024-02-22 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-02-06 | - |
| dc.identifier.citation | 鍾孟儒 (2021). 水稻經過氧化氫處理下Squamosa promoter-binding-like protein 2 (SPL2)的調控。國立台灣大學植物科學研究所碩士論文。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/91792 | - |
| dc.description.abstract | 自然環境中，植物無時無刻面對著各種生物或非生物性逆境，並啟動防禦機制以作出應對。當面臨外界壓力時，植物體內會產生活性氧化物（Reactive Oxygen Species，ROS），如：過氧化氫（Hydrogen peroxide，H2O2）與氫氧自由基（Hydroxyl radical，OH•）等。過多的ROS會使植物面臨氧化逆境；然而，ROS也同時可作為二次訊息分子，啟動下游基因調控。實驗室先前研究主要關注在H2O2處理所導致的氧化逆境下，14天大水稻幼苗根部的訊息傳遞。實驗室前人建立了三筆水稻資料庫：small RNA deep sequencing data、degradome及transcriptome。利用這些資料並加以驗證，發現H2O2的處理在短時間內降低了microRNA156（miR156）的表達量，並導致其目標基因SQUAMOSA promoter-binding-like protein 2（OsSPL2）的上調。此外，OsSPL2會作為負調控者，抑制其下游基因Heme Activator Protein 2J（OsHAP2J）的表現。OsHAP2J又被稱為nuclear factor YA5 （OsNF-YA5），屬於nuclear factor Y（NF-Y）家族。NF-YA（YA）與NF-YB（YB）和NF-YC（YC）會形成三聚體(heterotrimer)，並通過YA的DNA結合域，結合下游基因啟動子中的CCAAT-box來加以調控。本研究的目標是找到OsHAP2J之調控模型與其互動蛋白，YB與YC，並通過建立和交叉比較各轉錄體資料庫來探索OsHAP2J的目標基因。藉由這些研究策略發現，OsHAP2J可能抑制一個具有未知功能域（domain of unknown function，DUF）DUF1719的蛋白基因Os07g0122000，且該基因可能與水稻的抗病機制有關。綜合這些結果，推測在H2O2處理下，miR156所調控的下游途徑會影響水稻的抗病性。 | zh_TW |
| dc.description.abstract | Plants respond to biotic or abiotic stresses all the time. When facing stresses, plants produce reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and hydroxyl radical (HO•). Excessive ROS causes plants to encounter oxidative stress, but at the same time, ROS can also serve as a secondary signal to initiate downstream regulations. Our previous studies mainly focused on the signaling pathway within 14-day-old rice root under H2O2-treated oxidative stress. Several databases, including small RNA deep sequencing, degradome, and transcriptome, were built in rice. Based on these databases and further validation, the expression of microRNA 156 (miR156) was decreased in rice treated with H2O2, and caused the upregulation of miR156’s target gene, SQUAMOSA promoter-binding-like protein 2 (OsSPL2). In addition, OsSPL2, acting as a negative regulator, suppresses the expression of its downstream gene, Heme Activator Protein 2J (OsHAP2J). OsHAP2J is a nuclear factor YA5 (OsNF-YA5) that belongs to the Nuclear Factor Y (NF-Y) family. NF-YA (YA) forms a heterotrimer with NF-YB (YB) and NF-YC (YC), and binds to the CCAAT-box of downstream genes through the DNA binding domain of YA. Here, the regulatory module of OsHAP2J was investigated and its interaction partners, YB and YC, was screened by yeast-two hybridization. This study suggested that OsHAP2J may suppress the expression of Os07g0122000 encoding a domain called DUF1719, and Os07g0122000 may be associated with disease resistance in rice. Hence, we hypothesized that the molecular mechanism regulated by miR156 affects the disease resistance of rice under H2O2 treatment. | en |
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| dc.description.tableofcontents | 序言 i
中文摘要 ii Abstract iii Content iv Introduction 1 1. Rice and plant stresses 1 1.1 Rice 1 1.2 Plant stress responses 1 2. Reactive oxygen species and oxidative stress 3 2.1 ROS and hydrogen peroxide, H2O2 3 2.2 Oxidative stress 3 3. The miR156/SQUAMOSA promoter binding protein-like (SPL) module in plants 5 3.1 Biogenesis and regulation of microRNA (miRNA) 5 3.2 miR156 and OsSPL2 6 4. Nuclear factor Y (NF-Y) family 7 5. Domains of Unknown Function (DUFs) 10 6. Research motivation and objectives 10 Materials and Methods 12 1. Plant materials 12 1.1 Wildtype rice 12 1.2 Transgenic rice 12 2. H2O2 treatment 13 3. Agrobacterium preparation and rice stable transformation 14 3.1 Agrobacterium competent cell preparation 14 3.2 Agrobacterium electroporation 14 3.3 Rice stable transformation 15 3.4 CRISPR/cas9 17 4. DNA and RNA manipulation 17 4.1 Plant genomic DNA extraction 17 4.2 Plant total RNA extraction 18 4.3 DNase treatment and mRNA reverse transcription 19 4.4 Polymerase chain reaction (PCR) and quantitative real-time PCR (RT-qPCR) 19 4.5 Gel electrophoresis 20 4.6 Genotyping 21 5. RNA sequencing 21 6. Plasmid construction 22 6.1 Gene cloning 22 6.2 Gateway LR recombination 22 6.3 Restriction enzyme 23 6.4 Gel purification, PCR product and enzyme restriction cleanup 23 6.5 DNA ligation 23 6.6 Bacteria transformation 24 6.7 Plasmid DNA extraction 24 7. Promoter analysis 25 8. Yeast two-hybrid (Y2H) assay 25 8.1 Yeast competent cell preparation 25 8.2 Yeast transformation 26 8.3 Yeast mating and screening (small-scale) 27 8.4 Yeast mating and screening (library-scale) 27 9. Bimolecular fluorescence complementation (BiFC) assay 28 9.1 Plasmid DNA extraction (NucleoBond Xtra Midi) 28 9.2 Rice protoplast isolation 29 9.3 Rice protoplast transformation 30 9.4 BiFC assay and visualization under confocal microscopy 32 Results 33 1. Generation of OsHAP2J transgenic lines 33 2. Analysis of transcriptome data from transgenic and wildtype rice 34 3. The function of OsHAP2J analyzed by Gene Set Enrichment Analysis (GSEA) 35 4. OsHAP2J negatively regulates Os07g0122000 37 5. Protein structural analysis of Os07g0122000 38 6. OsHAP2J physically interacts with OsNF-YC4 39 7. Detection of OsHAP2J, OsNF-YB11, and OsNF-YC4 co-expression under oxidative stress 41 Discussion 43 1. The potential roles and functions of OsHAP2J in rice 43 2. OsHAP2J may collaborate with OsNF-YB11 and OsNF-YC4, forming a heterotrimeric complex 45 3. OsNF-Ys in rice under H2O2 treatment 46 4. Os07g0122000 encodes an unknown function protein, DUF1719 48 5. H2O2 in rice stress responses 49 6. Conclusion 50 Tables 52 Table 1. Primers used in this study. 52 Table 2. Prediction of genes regulated by OsHAP2J. 54 Table 3. Prediction of genes regulated by OsHAP2J using GSEA. 55 Table 4. Results of Y2H library screening. 56 Table 5. Expression of NF-Ys under H2O2 treatment. 57 Figures 58 Figure 1. Construction and verification of OsHAP2J mutant M0093930. 58 Figure 2. Verification and phenotype of OsHAP2J mutant M0093930. 59 Figure 3. RNA-seq results of OsHAP2J T-DNA insertion mutant M930. 60 Figure 4. Gene Set Enrichment analysis (GSEA) of HAP2J T-DNA insertion mutant M930. 61 Figure 5. Promoter analysis and gene expression of Os07g0122000. 62 Figure 6. Protein structure analysis of Os07g0122000. 63 Figure 7. Construction and expression of Os07g0122000 transgenic rice M0102411. 64 Figure 8. Verification of the interaction between OsHAP2J and OsNF-YC4. 65 Figure 9. Verification of the interaction between OsHAP2J and OsNF-YC4. 66 Figure 10. Expression levels of OsNF-YB11 and OsNF-YC4 under H2O2 treatment. 67 Figure 11. Regulation of OsHAP2J in rice under H2O2 treatment. 68 Supplementary Data 69 Supplementary Figure S1. Regeneration and verification of 35S::OsHAP2J overexpression lines. 69 Supplementary Figure S2. Regeneration and verification of CRISPR/cas9 OsHAP2J knockout lines. 70 Supplementary Figure S3. Expression levels of OsHAP2J’s candidate target genes by RT-qPCR assays. 71 Supplementary Figure S4. Expression levels of OsHAP2J’s candidate target genes. 72 Supplementary Figure S5. Expression level of OsHAP2J in rice under H2O2 treatment (Chung, 2021). 73 Supplementary Figure S6. Structural predictions and analysis of Os07g0122000. 74 Supplementary Figure S7. Expression levels of OsHAP2J’s candidate target genes. 75 Supplementary Figure S8. OsHAP2J overexpression vector in rice stable transformation. 76 Supplementary Figure S9. OsHAP2J knockout vector in CRISPR/Cas9. 77 Supplementary Figure S10. BD-OsHAP2J vector in Y2H assay. 78 Supplementary Figure S11. AD-OsNF-YC4 vector in Y2H assay. 79 Supplementary Figure S12. H2A-mCherry vector in BiFC assay. 80 Supplementary Figure S13. OsHAP2J-nYFP vector in BiFC assay. 81 Supplementary Figure S14. OsNF-YC4-cYFP vector in BiFC assay. 82 References 83 | - |
| dc.language.iso | en | - |
| dc.subject | 水稻 | zh_TW |
| dc.subject | 氧化逆境 | zh_TW |
| dc.subject | miR156 | zh_TW |
| dc.subject | NF-Ys | zh_TW |
| dc.subject | OsHAP2J | zh_TW |
| dc.subject | DUF1719 | zh_TW |
| dc.subject | 抗病機制 | zh_TW |
| dc.subject | oxidative stress | en |
| dc.subject | rice | en |
| dc.subject | disease response | en |
| dc.subject | DUF1719 | en |
| dc.subject | OsHAP2J | en |
| dc.subject | NF-Ys | en |
| dc.subject | miR156 | en |
| dc.title | 水稻在過氧化氫處理下HEME ACTIVATOR PROTEIN 2J（OsHAP2J）對Os07g0122000的調控 | zh_TW |
| dc.title | Regulation of Os07g0122000 by HEME ACTIVATOR PROTEIN 2J (OsHAP2J) in rice (Oryza sativa) under hydrogen peroxide treatment | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-1 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 靳宗洛;楊淑怡;林振祥 | zh_TW |
| dc.contributor.oralexamcommittee | Tsung-Luo Jinn;Shu-Yi Yang;Jeng-Shane Lin | en |
| dc.subject.keyword | 水稻,氧化逆境,miR156,NF-Ys,OsHAP2J,DUF1719,抗病機制, | zh_TW |
| dc.subject.keyword | rice,oxidative stress,miR156,NF-Ys,OsHAP2J,DUF1719,disease response, | en |
| dc.relation.page | 95 | - |
| dc.identifier.doi | 10.6342/NTU202400514 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2024-02-06 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 植物科學研究所 | - |
| 顯示於系所單位： | 植物科學研究所 | |
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