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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉嚞睿(Je-Ruei Liu) | |
| dc.contributor.author | Qian-Wen Shang | en |
| dc.contributor.author | 尚芊彣 | zh_TW |
| dc.date.accessioned | 2021-06-16T03:41:31Z | - |
| dc.date.available | 2020-08-25 | |
| dc.date.copyright | 2020-08-25 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54917 | - |
| dc.description.abstract | 本研究第一部分應用CRISPR/Cas9方法創造基因剔除之阿拉伯芥 (Arabidopsis thaliana)，探討基因功能上差異。轉錄後基因靜默作用 (post-transcriptional gene silencing, PTGS) 是植物應用序列特異性以降解或抑制轉譯來達到調控RNA的系統，當系統中RISC複合體 (RNA-induced silencing complex) 所載運小片段RNA (small RNA) 與目標RNA互補配對，Argonaute1 (AGO1) 蛋白即負責降解目標RNA。自噬作用 (autophagy) 負責生物體內細胞物質回收，降解不再需要的物質、轉化為新的所需形式。進行自噬作用包含許多相關蛋白，其中ATG8a及ATG10為兩個作用在不同路徑之蛋白。另一方面，HC-Pro為一Potyvirus病毒基因靜默抑制子，會破壞微型核酸 (miRNA) 生合成路徑以干擾轉錄後基因靜默作用。然而確切激發及相互作用途徑則尚未研究透徹。本篇研究顯示在HC-Pro所誘導之基因靜默抑制現象中，AGO1受到降解的現象與ATG8a至乎相關。同時，我們發現ATG8a是維持轉基因穩定表現的重要基因之一。實驗結果顯示CRISPR/Cas9編輯之atg8a突變株在P1/HCR轉基因植物具有與Col-0相當的AGO1蛋白含量。而ATG8a缺失造成HC-Pro在若干個別植株中有不穩定的表現。本研究結果展示AGO1如何在P1/HCR植株中被抑制及ATG8a在支持PTGS所扮演的角色。 延續第一部分的研究應用CRISPR/Cas9系統的經驗，我們同時觀察到近年來使用CRISPR/Cas9系統進行基因編輯的物種日益增多，其大多來自化膿鏈球菌 (Streptococcus pyogenes)。因此，本研究第二部分針對一株具有益生潛力的洛德乳桿菌 (Lactobacillus reuteri Pg4) 進行全基因體序列分析，找出其Cas9、Cas1及Cas2基因，此發現有助於將Pg4菌株開發為適用於乳酸桿菌屬之基因編輯工具。 | zh_TW |
| dc.description.abstract | In the first part of the study, we applied CRISPR/Cas9 system to knock-out genes in Arabidopsis and studied the significance of the gene functions. Post-transcriptional gene silencing (PTGS) is a system which regulates RNA levels by targeted degradation or translation inhibition. Argonaute1 (AGO1) in the RNA-induced silencing complex (RISC) is responsible for the cleavage of the targets when small RNAs (sRNAs) in this complex pair with the target RNAs complementarily. Autophagy, in addition, is a dynamic recycling system for cellular components, degrading unwanted substance for remodeling into a new form as needed. The system consists of several autophagy-related (ATG) proteins, in which ATG8a and ATG10 work independently in the pathway. On the other hand, HC-Pro, the viral silencing suppressor (VSR) from potyvirus, was known to interfere PTGS by collapsing miRNA biosynthesis. However, the specific triggering and interacting pathway are yet to be thoroughly confirmed. Here we showed that ATG8a plays a key role in AGO1 degradation in HC-Pro-mediated silencing suppression and that ATG8a stabilizes transgene expression. We found that CRISPR/Cas9-edited atg8a mutant in P1/HCR plant had comparable AGO1 expression with that of Col-0. The absence of ATG8a in P1/HCR plant caused inconsistent expression of HC-Pro among some individual lines. Our results exemplify how AGO1 is suppressed in P1/HCR context and what role ATG8a plays in supporting PTGS. With the experience of applying CRISPR/Cas9 system in the first part, we observed that CRISPR/Cas9 system is widely adopted as genome-editing tools and the majority comes from that of Streptococcus pyogenes. In the second part of this study, we examined the whole genome sequence of a potential probiotic, Lactobacillus reuteri Pg4 and identified Cas9 along with Cas1 and Cas2. This finding may help the Pg4 strain be engineered into a genome-editing tool tailored for the genus Lactobacillus. | en |
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| dc.description.tableofcontents | 誌謝 I 中文摘要 II Abstract III Contents IV List of Tables VI List of Figures VII Chapter 1. Investigation of the role of ATG genes in PTGS suppression through CRISPR/Cas9 knock out approach 1 Introduction 2 PTGS defense and P1/HC-Pro silencing suppression 2 Autophagy and its related genes ATG8a and ATG10 4 CRISPR/Cas9 application in genome editing 5 Materials and Methods 8 Plant materials and growth conditions 8 ATG8a and ATG10 genes editing with CRISPR/Cas9 8 Genotyping 9 Protein degradation inhibiting assay 9 Cucumber mosaic virus (CMV) Q strain inoculation 9 RNA extraction and real-time RT-PCR 10 Protein preparation and western blotting 10 ABA sensitivity assay 11 Germination assay in starvation 11 Results 12 ATG8a and ATG10 knock out in P1/HCR and Col-0 background by CRISPR/Cas9 gene editing technology 12 The phenotype of atg mutants in Col-0 and P1/HCR backgrounds 14 P1/HCR plant and atg8age mutant are ABA sensitive 15 Mimic autophagy initiation in the absence of light and carbon source 17 Epigenetically-regulated phenotypic variation in T3 generation of P1/HCR/atg8age plants 18 HC-Pro reducing in atg8age background 19 HC-Pro stable expression in P1/HCK plant 20 Discussion 22 Multiple gRNAs providing high efficiency of gene editing 22 ATG8a involves in autophagic AGO1 degradation and stabilizing the transgene expression 22 Working hypotheses 25 Conclusions 26 References 27 Chapter 2. Identification of Cas genes in Lactobacillus reuteri Pg4 41 Introduction 42 Cas genes in Lactobacillus spp. 42 Lactobacillus reuteri Pg4 and lactic acid bacteria 43 Genome editing in bacteria with CRISPR/Cas system 44 Materials and Methods 46 Bacterial strain and growth conditions 46 Whole genome sequencing (WGS) and genome analysis 46 Cas9-related analysis 47 Results 49 Whole genome sequence of L. reuteri Pg4 49 Cas genes in Pg4 50 Discussion 53 A unique Cas9 gene in Pg4 53 Conclusion 55 Reference 56 | |
| dc.language.iso | en | |
| dc.subject | CRISPR/Cas9 | zh_TW |
| dc.subject | 轉錄後基因靜默作用 | zh_TW |
| dc.subject | 基因靜默抑制子 | zh_TW |
| dc.subject | P1/HC-Pro | zh_TW |
| dc.subject | 自噬作用 | zh_TW |
| dc.subject | 乳酸桿菌 | zh_TW |
| dc.subject | Post-transcriptional gene silencing | en |
| dc.subject | CRISPR/Cas9 | en |
| dc.subject | Lactobacilli | en |
| dc.subject | Autophagy | en |
| dc.subject | P1/HC-Pro | en |
| dc.subject | Gene silencing suppressor | en |
| dc.title | 鑑別Lactobacillus reuteri Pg4中Cas9基因及利用CRISPR/Cas9基因剔除方法研究ATG基因在抑制轉錄後基因靜默作用中所扮演的角色 | zh_TW |
| dc.title | Identification of Cas9 gene in Lactobacillus reuteri Pg4 and investigation of the role of ATG genes in PTGS suppression through CRISPR/Cas9 knock out approach | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 林詩舜(Shih-Shun Lin) | |
| dc.contributor.oralexamcommittee | 吳素幸(Shu-Hsing Wu),林崇熙(Choun-Sea Lin),林劭品(Shau-Ping Lin) | |
| dc.subject.keyword | 轉錄後基因靜默作用,基因靜默抑制子,P1/HC-Pro,自噬作用,乳酸桿菌,CRISPR/Cas9, | zh_TW |
| dc.subject.keyword | Post-transcriptional gene silencing,Gene silencing suppressor,,P1/HC-Pro,Autophagy,Lactobacilli,CRISPR/Cas9, | en |
| dc.relation.page | 68 | |
| dc.identifier.doi | 10.6342/NTU202002193 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-04 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 生物科技研究所 | zh_TW |
| 顯示於系所單位： | 生物科技研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| U0001-0108202010445100.pdf 未授權公開取用 | 14.77 MB | Adobe PDF |
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