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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/94432完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭石通 | zh_TW |
| dc.contributor.advisor | Shih-Tong Jeng | en |
| dc.contributor.author | 洪育翎 | zh_TW |
| dc.contributor.author | Yu-Ling Hung | en |
| dc.date.accessioned | 2024-08-15T17:28:00Z | - |
| dc.date.available | 2024-08-16 | - |
| dc.date.copyright | 2024-08-15 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-08-08 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/94432 | - |
| dc.description.abstract | 植物的微型核酸 (miRNAs) 在後轉錄層級調控目標基因的表現，控制著植物生長發育的過程。其中，保守性的miRNAs在植物發育的所有方面幾乎都發揮了至關重要的作用。本論文的研究展示保守的miR390在基礎陸地植物地錢中的分子調控機制及功能並建立了開花植物中花瓣原生質體暫時性轉殖系統，為研究miR390調控開花相關基因提供了一個技術平台。在第二章中，研究顯示在地錢中miR390透過裁切TAS3轉錄本產生轉導性短干擾RNAs (tasiRNAs)，從中找到一個大量表現的tasiRNA，tasi78A，並預測到其目標基因為一個細胞色素P450基因，MpCYP78A101。同時還預測到MpCYP78A101受到另一個miRNA, miR11700共同調控。透過暫時性轉殖系統驗證了tasi78A及miR11700負向調控MpCYP78A101，並且可能通過調控生長素訊息來影響地錢的無性生殖器官孢芽 (gemma) 以及有性生殖器官的生長發育。第三章則是建立了開花植物苦苣苔屬中大岩桐的花瓣原生質體暫時性轉殖系統，將切成條狀的花瓣浸泡在1.5%纖維素酶 (cellulase) 和 0.4% 離析酶 (macerozyme) 的酵素溶液中六小時，可獲得高產量的花瓣原生質體，並達到41.4%的轉殖效率。本論文對於保守的miR390從基礎陸地植物到開花植物在演化上、基因調控機制及功能性將有更全面的了解。 | zh_TW |
| dc.description.abstract | Plant microRNAs (miRNAs) regulate target gene expression at the post-transcriptional level and control the growth and development of plants. Among them, conserved miRNAs play key roles in plant development, with functions spanning almost all aspects. This study unveils the molecular regulatory mechanisms and functions of the conserved miR390 in the basal land plant, Marchantia polymorpha. Moreover, a petal protoplast transient transformation system in flowering plant was established, providing a technical platform for investigating miR390 regulation of flowering-related genes. In Chapter 2, we demonstrated that miR390 generates trans-acting short-interfering RNAs (tasiRNAs) by cleaving TAS3 transcripts in M. polymorpha. Among these tasiRNAs, one highly expressed tasiRNA, tasi78A, was identified, and its target gene was predicted to be a cytochrome P450 gene, MpCYP78A101. Moreover, we further predicted that MpCYP78A101 is co-regulated by another miRNA, miR11700. Using a transient transformation system, we validated that tasi78A and miR11700 negatively regulate MpCYP78A101 and potentially affect the development of asexual reproductive organs (gemmae) and sexual organs through auxin signaling. In Chapter 3, we established a petal protoplast transient transformation system in the flowering plant Gloxinia (Sinningia speciosa). By immersing petal strips in an enzyme solution containing 1.5% cellulase and 0.4% macerozyme for 6 hours, we obtained high yields of petal protoplasts and achieved a transformation efficiency of 41.4%. This study provides a further understanding of the evolution, gene regulatory mechanisms, and functional roles of conserved miR390 from basal land plants to flowering plants. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-08-15T17:28:00Z No. of bitstreams: 0 | en |
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| dc.description.tableofcontents | 致謝 I
摘要 II Abstract III Content IV Chapter 1 Literature review 1 1.1 Introduction 2 1.2 RNA silencing 2 1.2.1 Biogenesis and types of small RNAs 2 1.2.2 The conservation of miRNA 3 1.3 The knowledge of miR390 4 1.3.1 miR390 specifically loads into AGO7 4 1.3.2 The regulatory mechanism and function of miR390 4 1.3.3 The conservation of miR390/TAS3 module 5 1.4 The miR390/TAS3 module in bryophytes 6 1.4.1 The possible diverse function for miR390/TAS3 module in bryophytes 6 1.4.2 Advantages of studying M. polymorpha 7 1.4.3 Recent study of small RNA-mediated gene silencing pathway and miR390/TAS3 module in M. polymorpha 8 1.5 MiR390/TAS3 module in floral symmetry 11 1.5.1 Recent study of miR390/TAS3 module in Sinningia speciosa 11 1.5.2 Transient transformation system 12 1.6 Aim of this study 14 Chapter 2 Dual regulation of cytochrome P450 gene expression by two distinct small RNAs, a novel tasiRNA and miRNA, in Marchantia polymorpha. 16 2.1 Abstract 17 2.2 Introduction 18 2.3 Materials and Methods 21 2.3.1 Plant materials and growth conditions 21 2.3.2 Gene construction 21 2.3.3 Plant transformation and gene knock-out 22 2.3.4 Small RNA detection 22 2.3.5 MpAGO1-IP 23 2.3.6 Small RNA deep sequencing 23 2.3.7 Promoter-reporter assays 24 2.3.8 Small RNA target identification 24 2.3.9 Real-time RT–PCR and stem-loop qRT-PCR 25 2.3.10 Transient expression by agroinfiltration 26 2.3.11 Transient expression by agropenetration in M. polymorpha 26 2.3.12 Auxin sensitivity assay 27 2.3.13 Cell size quantification 28 2.3.14 Phylogenetic tree of CYP78 and CYP98 families 28 2.4 Results 28 2.4.1 miR390/TAS3/MpARF2 pathway in M. polymorpha 28 2.4.2 MpTAS3-derived tasiRNAs are mainly processed from the 3'-end phasing positions 1 and 2 of MpTAS3 transcripts 30 2.4.3 A novel tasi78A regulates MpCYP78A101 31 2.4.4 miR390 leads to the production of tasiRNAs in M. polymorpha 32 2.4.5 MpCYP78A101 was regulated by both tasi78A and miR11700 33 2.4.6 Promoter assay for MIR390, MpTAS3, MIR11700, and MpCYP78A101 34 2.4.7 Tasi78A negatively regulates MpCYP78A101 in M. polymorpha 35 2.4.8 miR11700 may regulate MpCYP78A101 at the reproductive stage in M. polymorpha 35 2.4.9 Evaluation of the regulatory relationships between tasi78A, miR11700, and MpCYP78A101 in M. polymorpha 37 2.4.10 The dcl4ge, mir390ge, and mir390/11700ge mutants display NAA less sensitive phenotype 38 2.4.11 Target sites of tasi78A and miR11700 in CYP78 homologs during land plant evolution 39 2.5 Discussion 40 2.6 Conclusion 46 2.7 Tables and Figures 47 Figure 1. Target prediction of miR390 and tasiARF and the role of MpDCL4 in tasiRNA biogenesis in M. polymorpha. 47 Figure 2. MpTAS3-derived phased 21-nucleotide (nt) tasiRNAs and their corresponding targets in M. polymorpha. 49 Figure 3. Small RNA expression patterns of miR390, tasiARF, and tasi78A in the mir390ge mutants and MIR390OE plants. 51 Figure 4. Target prediction of mi11700 and the reporter assays of amiR-tasi78A, miR11700, and MpCYP78A101 in Nicotiana benthamiana. 52 Figure 5. Expression patterns of the MIR390/MpTAS3 module, MIR11700, and MpCYP78A101 in M. polymorpha. 54 Figure 6. Phenotypic observations and reporter assays of 78ARepYFP in the amiR-TASI78AOE plants. 56 Figure 7. Phenotypic observations and expression analysis of the mir11700ge mutants and MIR11700OE plants. 57 Figure 8. Phenotypic observations and reporter assays of 11700RepYFP in the mir390/11700ge mutants and MIR390/11700OE plants. 59 Figure 9. NAA treatment of dcl4ge, mir390ge, mir390/11700ge mutants, and amiR-TASI78AOE plants. 61 Figure 10. Phylogenetic tree of CYP78 homologs. 63 Figure 11. Illustration of the miR390/TAS3 regulatory module in Arabidopsis and M. polymorpha. 64 2.8 Supplementary Tables and Figures 65 Supplementary Table S1. The primer sets used for gene construction in this study 65 Supplementary Figure S1. MpTAS3-derived phased 21-nucleotide tasiRNAs. 67 Supplementary Figure S2. Alignments and target plots of tasi78A and its targets in M. polymorpha. 68 Supplementary Figure S3. Alignments and target plots of tasiRNAs from 3'-end phasing position 2 and the targets in M. polymorpha. 73 Supplementary Figure S4. Boxplot of quantified YFP intensity of the confocal images. 74 Supplementary Figure S5. Phenotypic observations and expressional analysis of the mir390ge mutants and MIR390 plants. 75 Supplementary Figure S6. Boxplot of thallus width and sexual organ number of mir11700ge mutants and MIR11700 plants at reproductive stage. 76 Supplementary Figure S7. qRT-PCR of MpCYP78A101 in the mir390/11700ge mutants by using 2-week-old thallus. 77 Supplementary Figure S8. Boxplot of the area of NAA-treated thallus. 78 Supplementary Figure S9. Alignments of tasi78A and CYP78 homologs. 79 Supplementary Figure S10. Alignments of miR11700 and CYP78 homologs. 80 2.9 Appendix 81 Appendix 1. CRISPR/Cas9-mediated mutation on MpDCL4. 81 Appendix 2. Expression patterns of MIR11700 and MpCYP78A101 in the male sexual organs of M. polymorpha. 83 Appendix 3. Phenotypic observations of dcl4ge, mir390ge, mir390/11700ge mutants, and amiR-TASI78AOE plants 84 Appendix 4. NAA treatment of dcl4ge, mir390ge, mir390/11700ge mutants, and amiR-TASI78AOE plants. 85 Chapter 3 Development of a petal protoplast transfection system for Sinningia speciosa 86 3.1 Abstract 87 3.2 Introduction 88 3.3 Material and Methods 90 3.3.1 Plant growth conditions 90 3.3.2 Petal protoplast isolation 90 3.3.3 DNA transfection 91 3.4 Results 92 3.4.1 Petal protoplast isolation and protoplast yield 92 3.4.2 DNA transformation into petal protoplasts form S. speciosa 93 3.5 Discussion 95 3.5.1 Moderate yields and transfection efficiency of S. speciosa petal protoplasts 95 3.6 Tables and Figures 97 Table 1 Comparison of protoplast isolation condition in different species. 97 Figure 1 Effect of osmotic pressure (mannitol concentration) on protoplast yield from petal in S. speciosa. 99 Figure 2 Transfection efficiency of transfected Sinningia speciosa ‘ES’ petal protoplasts. 100 3.7 Appendix 101 Appendix 1. Reagents and Equipment required to perform the isolation and transfection of S. speciosa petal protoplasts. 101 Reference 103 Appendix 115 | - |
| dc.language.iso | en | - |
| dc.subject | miR390/TAS3 | zh_TW |
| dc.subject | tasiRNA 生合成 | zh_TW |
| dc.subject | 生長素訊息 | zh_TW |
| dc.subject | MpCYP78A101 | zh_TW |
| dc.subject | 地錢 | zh_TW |
| dc.subject | 大岩桐 | zh_TW |
| dc.subject | 原生質體 | zh_TW |
| dc.subject | MpCYP78A101 | en |
| dc.subject | tasiRNA biogenesis | en |
| dc.subject | auxin signaling | en |
| dc.subject | protoplast | en |
| dc.subject | Sinningia speciosa | en |
| dc.subject | Marchantia polymorpha | en |
| dc.subject | miR390/TAS3 | en |
| dc.title | 地錢細胞色素P450受tasiRNA及微型核酸雙調控機制研究及大岩桐花瓣原生質體暫時性表達系統之建立 | zh_TW |
| dc.title | Dual regulation of cytochrome P450 expression by tasiRNA and microRNA in Marchantia polymorpha and the establishment of Sinningia petal protoplast transient expression system | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-2 | - |
| dc.description.degree | 博士 | - |
| dc.contributor.coadvisor | 林詩舜;王俊能 | zh_TW |
| dc.contributor.coadvisor | Shih-Shun Lin;Chun-Neng Wang | en |
| dc.contributor.oralexamcommittee | 西浜竜一;小松愛乃;吳素幸;陳荷明;邱子珍 | zh_TW |
| dc.contributor.oralexamcommittee | Ryuichi Nishihama;Aino Komatsu;Shu-Hsing Wu;Ho-Ming Chen;Tzyy-Jen Chiou | en |
| dc.subject.keyword | miR390/TAS3,tasiRNA 生合成,生長素訊息,MpCYP78A101,地錢,大岩桐,原生質體, | zh_TW |
| dc.subject.keyword | miR390/TAS3,tasiRNA biogenesis,auxin signaling,MpCYP78A101,Marchantia polymorpha,Sinningia speciosa,protoplast, | en |
| dc.relation.page | 115 | - |
| dc.identifier.doi | 10.6342/NTU202404007 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2024-08-12 | - |
| 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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