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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 詹世鵬(Shih-Peng Chan) | |
| dc.contributor.author | Cheng-Fu Tseng | en |
| dc.contributor.author | 曾鉦富 | zh_TW |
| dc.date.accessioned | 2022-11-25T06:34:56Z | - |
| dc.date.copyright | 2021-11-09 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-28 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82282 | - |
| dc.description.abstract | 微小核醣核酸(MicroRNAs)是一段長約22個核甘酸的非編碼RNA，其作用方式為透過miRISC complex結合上目標mRNA的3′端不轉譯區，抑制目標基因的轉譯或是使目標基因降解。其中，let-7為最早被發現的miRNA之一，在線蟲中負責調控第四幼蟲期至成蟲的過渡階段，低量的let-7表現會無法有效抑制下游目標lin-41，而造成線蟲的上皮接縫細胞(hypodermal seam cell)終端分化遲緩以及外陰(vulva)發育異常。具let-7點突變之線蟲let-7(n2853)在第四幼蟲期的末期會發生上皮接縫細胞異常多分裂一次以及外陰爆裂的性狀。而在不同物種間皆發現有let-7與lin-41的路徑，顯示這是一個具演化保守性的發育調節機制，且let-7的缺失已被發現與許多癌症相關。為了找出可能參與let-7生合成或功能有關之異質核糖核蛋白(hnRNPs)或DEAD-box核糖核酸解旋酶蛋白，實驗室先前已利用RNAi篩選出MSI-1、DDX-47以及hnRNP L幾個候選蛋白，但在我們進一步實驗以後，發現MSI-1以及DDX-47可能不參與在let-7路徑。降低DDX-47僅會延遲線蟲生長並不會降低上皮接縫細胞的數目，而降低hnRNP L表現可以降低n2853線蟲的上皮接縫細胞數，且不會抑制線蟲的生長。利用GFP後接lin-41 3′ UTR以及mCherry後接unc-54 3′ UTR的雙色螢光報告基因之線蟲探討hnRNP L與let-7關係，發現降低hnRNP L不只不會下降雙色螢光報告基因線蟲lin-41 3′ UTR的GFP，反而上升。同時，原本預期不會影響有剔除let-7結合位點之雙色螢光報告基因線蟲的lin-41 3′ UTR，結果也看到GFP上升，而我們北方墨點法以及即時聚合酶連鎖反應的結果皆顯示降低hnRNP L並不會影響mature let-7的生成量，綜合以上這些結果，顯示降低hnRNP L可能不只會影響到let-7一個路徑，而是會透過影響其他相關路徑，進而影響到線蟲lin-41表現以及上皮接縫細胞的發育，至於hnRNP L會如何影響lin-41的表現以及與let-7下游lin-29之間的關係，仍有待未來進一步的探討。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T06:34:56Z (GMT). No. of bitstreams: 1 U0001-2709202117263800.pdf: 5415336 bytes, checksum: bf8d93677b1db7901d693a7e903f3f6f (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "口試委員審定書 i 中文摘要 ii ABSTRACT iii CONTENTS v Chapter 1 Introduction 1 1.1 Function and biogenesis of miRNAs 1 1.2 The differentiation of C. elegans mediated by let-7 miRNA 2 1.3 Regulation of let-7 biosynthesis 3 1.4 Heterogenous nuclear ribonucleoproteins (hnRNPs) 5 1.4.1 The hnRNPs family and their structural features 5 1.4.2 Link of hnRNPs to diseases 5 1.4.3 The miRNAs biosynthesis-related hnRNPs 6 1.5 DExD/H-box RNA helicases 7 1.5.1 Cellular functions of DExD/H-box RNA helicases 7 1.5.2 The miRNAs biosynthesis-related DExD/H-box RNA helicases 7 1.6 Project aim 8 Chapter 2 Materials and methods 10 2.1 Caenorhabditis elegans 10 2.1.1 Strains 10 2.1.2 Culture conditions 11 2.1.3 Synchronization of C. elegans 11 2.1.4 RNAi clones 12 2.1.5 Feeding RNAi 12 2.1.6 Genomic DNA extraction 13 2.1.7 RNA extraction 14 2.2 Western blotting analysis 14 2.2.1 Protein extraction 15 2.2.2 Gel preparation 15 2.2.3 Protein Gel Electrophoresis / Transfer / Antibody hybridization 15 2.3 Northern blotting analysis 16 2.3.1 Preparing isotope-labeled DNA probe 16 2.3.2 Urea gel preparation 17 2.3.3 Gel Electrophoresis and Transfer 18 2.3.4 Hybridization and Wash 18 2.3.5 Detection and Quantification 19 2.3.5 Probe secquence 19 2.4 Random Primimg qRT-PCR 19 2.4.1 rDNase I digestion 20 2.4.2 Reverse Transcription 20 2.4.3 qPCR 21 2.4.4 Primer sequence 22 2.5 TaqMan RT-qPCR 23 2.5.1 Reverse Transcription 23 2.5.2 PCR Amplification 24 2.6 PCR primers 24 2.6.1 RNAi plasmid construction 25 Chapter 3 Results 28 3.1 MSI-1 might not be involved in let-7 pathway 28 3.1.1 Knockdown of msi-1 does not inhibit the seam cell number increase phenotype caused by wIs51; let-7(n2853) 28 3.1.2 Knockdown of msi-1 does not inhibit the seam cell number increase phenotype caused by wIs79; let-7(n2853) 29 3.2 DDX47 might not be involved in let-7 pathway 30 3.2.1 Knockdown of ddx-47 does not inhibit the seam cell number increase phenotype caused by wIs79; let-7(n2853) and wIs51; let-7(n2853) 30 3.2.2 Knockdown of ddx-47 can repress the multivulva phenotype caused by let-60(n1046) gain-of-function mutation 30 3.3 The relationship between hnRNP L and let-7 pathway 31 3.3.1 Knockdown of hrpl-1 can inhibit the seam cell number increase phenotype caused by wIs51; let-7(n2853) 31 3.3.2 Knockdown of hrpl-1 cannot rescue the hyp-7 expression of col-19::gfp; let-7(n2853) 32 3.3.3 Knockdown of hrpl-1 increases the fluorescence expression of GFP controlled by lin-41 3′UTR at mid-L4 stage in chsIs002; let-7(n2853) and chsIs001; let-7(n2853) dual-color reporter system 32 3.3.4 Knockdown of hrpl-1 does not affect the expression of LIN-29A of HW1826; let-7(n2853) 33 Chapter 4 Discussion 34 4.1 The relationship between MSI-1 and let-7 is still unclear 34 4.2 DDX-47 might not be involved in the let-7 pathway 35 4.3 A reduction in seam cells can be achieved by knockdown of hrpl-1, but it appears to be related to pathways other than let-7 35 Chapter 5 Figures 38 Figure 1. 38 Figure 2. 41 Figure 3. 44 Figure 4. 49 Figure 5. 51 Figure 6. 53 Figure 7. 55 Figure 8. 63 Chapter 6 References 65 Appendix 69 Figure 1. 69 Figure 2. 70 Figure 3. 71 Figure 4. 72 Appendix 73 Table 1. The Ct value of Real-Time PCR, normalized by eft-2 73 Table 2. The quantification of fluorescence of chsIs001; let-7(n2853). The value listed below is GFP/mCherry 74 Table 3. The quantification of fluorescence of chsIs002; let-7(n2853). The value listed below is GFP/mCherry 90" | |
| dc.language.iso | en | |
| dc.subject | MSI-1 | zh_TW |
| dc.subject | DDX47 | zh_TW |
| dc.subject | hnRNP L | zh_TW |
| dc.subject | let-7 | zh_TW |
| dc.subject | 微小核醣核酸 | zh_TW |
| dc.subject | hnRNP L | en |
| dc.subject | microRNA | en |
| dc.subject | let-7 | en |
| dc.subject | MSI-1 | en |
| dc.subject | DDX47 | en |
| dc.title | 確定MSI-1和hnRNP L以及DDX47是否調控線蟲類幹細胞接縫細胞的分化 | zh_TW |
| dc.title | "Determine whether MSI-1, hnRNP L and DDX47 regulate differentiation of C. elegans stem cell-like seam cells" | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 顏伯勳(Hsin-Tsai Liu),蔡欣祐(Chih-Yang Tseng) | |
| dc.subject.keyword | 微小核醣核酸,let-7,MSI-1,DDX47,hnRNP L, | zh_TW |
| dc.subject.keyword | microRNA,let-7,MSI-1,DDX47,hnRNP L, | en |
| dc.relation.page | 100 | |
| dc.identifier.doi | 10.6342/NTU202103405 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2021-09-29 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2026-09-16 | - |
| 顯示於系所單位： | 微生物學科所 | |
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