Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/99133Full metadata record
| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 劉嚞睿 | zh_TW |
| dc.contributor.advisor | Je-Ruei Liu | en |
| dc.contributor.author | 唐雅榛 | zh_TW |
| dc.contributor.author | Ya-Jhen Tang | en |
| dc.date.accessioned | 2025-08-21T16:31:02Z | - |
| dc.date.available | 2025-08-22 | - |
| dc.date.copyright | 2025-08-21 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-08-04 | - |
| dc.identifier.citation | Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A. J., Bambrick, J., Bodenstein, S. W., Evans, D. A., Hung, C.-C., O’Neill, M., Reiman, D., Tunyasuvunakool, K., Wu, Z., Žemgulytė, A., Arvaniti, E., … Jumper, J. M. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 630(8016), 493–500. https://doi.org/10.1038/s41586-024-07487-w
Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D. A., & Horvath, P. (2007). CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science, 315(5819), 1709–1712. https://doi.org/10.1126/science.1138140 Bermudez-Brito, M., Plaza-Díaz, J., Muñoz-Quezada, S., Gómez-Llorente, C., & Gil, A. (2012). Probiotic Mechanisms of Action. Annals of Nutrition and Metabolism, 61(2), 160–174. https://doi.org/10.1159/000342079 Biswas, A., Gagnon ,Joshua N., Brouns ,Stan J.J., Fineran ,Peter C., & and Brown, C. M. (2013). CRISPRTarget: Bioinformatic prediction and analysis of crRNA targets. RNA Biology, 10(5), 817–827. https://doi.org/10.4161/rna.24046 Bolotin, A., Quinquis, B., Sorokin, A., & Ehrlich, S. D. (2005). Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology, 151(8), 2551–2561. https://doi.org/10.1099/mic.0.28048-0 Chen, Y., Xu, J., & Chen, Y. (2021). Regulation of Neurotransmitters by the Gut Microbiota and Effects on Cognition in Neurological Disorders. Nutrients, 13(6), 2099. https://doi.org/10.3390/nu13062099 Collado, M. C., Gueimonde, M., & Salminen, S. (2010). Chapter 23 - Probiotics in Adhesion of Pathogens: Mechanisms of Action. In R. R. Watson & V. R. Preedy (Eds.), Bioactive Foods in Promoting Health (pp. 353–370). Academic Press. https://doi.org/10.1016/B978-0-12-374938-3.00023-2 Crawley, A. B., Henriksen, E. D., Stout, E., Brandt, K., & Barrangou, R. (2018). Characterizing the activity of abundant, diverse and active CRISPR-Cas systems in lactobacilli. Scientific Reports, 8(1), 11544. https://doi.org/10.1038/s41598-018-29746-3 Crooks, G. E., Hon, G., Chandonia, J.-M., & Brenner, S. E. (2004). WebLogo: A Sequence Logo Generator. Genome Research, 14(6), 1188–1190. https://doi.org/10.1101/gr.849004 Cryan, J. F., O’Riordan, K. J., Cowan, C. S. M., Sandhu, K. V., Bastiaanssen, T. F. S., Boehme, M., Codagnone, M. G., Cussotto, S., Fulling, C., Golubeva, A. V., Guzzetta, K. E., Jaggar, M., Long-Smith, C. M., Lyte, J. M., Martin, J. A., Molinero-Perez, A., Moloney, G., Morelli, E., Morillas, E., … Dinan, T. G. (2019). The Microbiota-Gut-Brain Axis. Physiological Reviews, 99(4), 1877–2013. https://doi.org/10.1152/physrev.00018.2018 De Gregorio, P. R., Juárez Tomás, M. S., & Nader-Macías, M. E. F. (2016). Immunomodulation of Lactobacillus reuteri CRL1324 on Group B Streptococcus Vaginal Colonization in a Murine Experimental Model. American Journal of Reproductive Immunology, 75(1), 23–35. https://doi.org/10.1111/aji.12445 Dinan, T. G., & Cryan, J. F. (2017). The Microbiome-Gut-Brain Axis in Health and Disease. Gastroenterology Clinics of North America, 46(1), 77–89. https://doi.org/10.1016/j.gtc.2016.09.007 FAO. (2006). Probiotics in food: Health and nutritional properties and guidelines for evaluation. https://www.fao.org/3/a0512e/a0512e.pdf Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. (2012). Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 109(39), E2579–E2586. https://doi.org/10.1073/pnas.1208507109 Gu, S., Zhang, J., Li, L., & Zhong, J. (2022). Repurposing the Endogenous CRISPR-Cas9 System for High-Efficiency Genome Editing in Lacticaseibacillus paracasei. ACS Synthetic Biology, 11(12), 4031–4042. https://doi.org/10.1021/acssynbio.2c00374 Guo, M., Liu, H., Yu, Y., Zhu, X., Xie, H., Wei, C., Mei, C., Shi, Y., Zhou, N., Qin, K., & Li, W. (2023). Lactobacillus rhamnosus GG ameliorates osteoporosis in ovariectomized rats by regulating the Th17/Treg balance and gut microbiota structure. Gut Microbes, 15(1), 2190304. https://doi.org/10.1080/19490976.2023.2190304 Guo, Q., Yan, Y., Zhang, Z., Xu, B., Bangash, H. lqbal, Sui, X., Yang, Y., Zhou, Z., Zhao, S., & Peng, N. (2023). Developing the Limosilactobacillus reuteri Chassis through an Endogenous Programmable Endonuclease-Based Genome Editing Tool. ACS Synthetic Biology, 12(11), 3487–3496. https://doi.org/10.1021/acssynbio.3c00450 Hsieh, F.-C., Lan, C.-C. E., Huang, T.-Y., Chen, K.-W., Chai, C.-Y., Chen, W.-T., Fang, A.-H., Chen, Y.-H., & Wu, C.-S. (2016). Heat-killed and live Lactobacillus reuteri GMNL-263 exhibit similar effects on improving metabolic functions in high-fat diet-induced obese rats. Food & Function, 7(5), 2374–2388. https://doi.org/10.1039/C5FO01396H Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., & Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology, 169(12), 5429–5433. https://doi.org/10.1128/jb.169.12.5429-5433.1987 Jansen, Ruud., Embden, Jan. D. A. van, Gaastra, Wim., & Schouls, Leo. M. (2002). Identification of genes that are associated with DNA repeats in prokaryotes. Molecular Microbiology, 43(6), 1565–1575. https://doi.org/10.1046/j.1365-2958.2002.02839.x Jiang, J., Li, K., Xiao, Y., Zhong, A., Tang, J., Duan, Y., & Li, Z. (2023). Limosilactobacillus reuteri Regulating Intestinal Function: A Review. Fermentation, 9(1), Article 1. https://doi.org/10.3390/fermentation9010019 Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science, 337(6096), 816–821. https://doi.org/10.1126/science.1225829 Liu, J. R., Lai ,S.F., & and Yu, B. (2007). Evaluation of an intestinal Lactobacillus reuteri strain expressing rumen fungal xylanase as a probiotic for broiler chickens fed on a wheat-based diet. British Poultry Science, 48(4), 507–514. https://doi.org/10.1080/00071660701485034 Liu, J.-R., Yu, B., Lin, S.-H., Cheng, K.-J., & Chen, Y.-C. (2005). Direct cloning of a xylanase gene from the mixed genomic DNA of rumen fungi and its expression in intestinal Lactobacillus reuteri. FEMS Microbiology Letters, 251(2), 233–241. https://doi.org/10.1016/j.femsle.2005.08.008 Liu, L., Yang, D., Zhang, Z., Liu, T., Hu, G., He, M., Zhao, S., & Peng, N. (n.d.). High-Efficiency Genome Editing Based on Endogenous CRISPR-Cas System Enhances Cell Growth and Lactic Acid Production in Pediococcus acidilactici. Applied and Environmental Microbiology, 87(20), e00948-21. https://doi.org/10.1128/AEM.00948-21 Liu, Y., Tran, D. Q., Fatheree, N. Y., & Marc Rhoads, J. (2014). Lactobacillus reuteri DSM 17938 differentially modulates effector memory T cells and Foxp3+ regulatory T cells in a mouse model of necrotizing enterocolitis. American Journal of Physiology - Gastrointestinal and Liver Physiology, 307(2), G177–G186. https://doi.org/10.1152/ajpgi.00038.2014 Madeira, F., Madhusoodanan, N., Lee, J., Eusebi, A., Niewielska, A., Tivey, A. R. N., Lopez, R., & Butcher, S. (2024). The EMBL-EBI Job Dispatcher sequence analysis tools framework in 2024. Nucleic Acids Res, 52(W1), W521–W525. PubMed. https://doi.org/10.1093/nar/gkae241 Makarova, K. S., Wolf, Y. I., Iranzo, J., Shmakov, S. A., Alkhnbashi, O. S., Brouns, S. J. J., Charpentier, E., Cheng, D., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J. M., Scott, D., Shah, S. A., Siksnys, V., Terns, M. P., Venclovas, Č., White, M. F., Yakunin, A. F., … Koonin, E. V. (2020). Evolutionary classification of CRISPR–Cas systems: A burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org/10.1038/s41579-019-0299-x Markowiak-Kopeć, P., & Śliżewska, K. (2020). The Effect of Probiotics on the Production of Short-Chain Fatty Acids by Human Intestinal Microbiome. Nutrients, 12(4), 1107. https://doi.org/10.3390/nu12041107 Metchnikoff E. (1908). Chapter V. Lactic acid as inhibiting intestinal putrefaction. In: In The prolongation of life. Optimistic studies. (pp. 161–183). New York, London: G.P. Putnam’s Sons. https://www.gutenberg.org/files/51521/51521-h/51521-h.htm Mojica, F. J. M., Díez-Villaseñor, C., García-Martínez, J., & Almendros, C. (2009). Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology, 155(3), 733–740. https://doi.org/10.1099/mic.0.023960-0 Mojica, F. J. M., Díez-Villaseñor, C., García-Martínez, J., & Soria, E. (2005). Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements. Journal of Molecular Evolution, 60(2), 174–182. https://doi.org/10.1007/s00239-004-0046-3 Mojica, F. J. M., Díez‐Villaseñor, C., Soria, E., & Juez, G. (2002). Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Molecular Microbiology, 36(1), 244–246. https://doi.org/10.1046/j.1365-2958.2000.01838.x Mu, Q., Tavella, V. J., & Luo, X. M. (2018). Role of Lactobacillus reuteri in Human Health and Diseases. Frontiers in Microbiology, 9, 757. https://doi.org/10.3389/fmicb.2018.00757 Mu, Y., Zhang, C., Li, T., Jin, F.-J., Sung, Y.-J., Oh, H.-M., Lee, H.-G., & Jin, L. (2022). Development and Applications of CRISPR/Cas9-Based Genome Editing in Lactobacillus. International Journal of Molecular Sciences, 23(21), Article 21. https://doi.org/10.3390/ijms232112852 Nayfach, S., Bhatnagar, A., Novichkov, A., Estevam, G. O., Kim, N., Hill, E., Ruffolo, J. A., Silverstein, R., Gallagher, J., Kleinstiver, B., Meeske, A. J., Cameron, P., & Madani, A. (2025). Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data (p. 2025.01.06.631536). bioRxiv. https://doi.org/10.1101/2025.01.06.631536 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I., Dohmae, N., Ishitani, R., Zhang, F., & Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, 156(5), 935–949. https://doi.org/10.1016/j.cell.2014.02.001 Qi, C., Ding, M., Li, S., Zhou, Q., Li, D., Yu, R., & Sun, J. (2021). Sex-dependent modulation of immune development in mice by secretory IgA–coated Lactobacillus reuteri isolated from breast milk. Journal of Dairy Science, 104(4), 3863–3875. https://doi.org/10.3168/jds.2020-19437 Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., Koonin, E. V., Sharp, P. A., & Zhang, F. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature, 520(7546), 186–191. https://doi.org/10.1038/nature14299 Robert, X., & Gouet, P. (2014). Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research, 42(W1), W320–W324. https://doi.org/10.1093/nar/gku316 Russel, J., Pinilla-Redondo, R., Mayo-Muñoz, D., Shah, S. A., & Sørensen, S. J. (2020). CRISPRCasTyper: An automated tool for the identification, annotation and classification of CRISPR-Cas loci (p. 2020.05.15.097824). bioRxiv. https://doi.org/10.1101/2020.05.15.097824 Sapranauskas, R., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath, P., & Siksnys, V. (2011). The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Research, 39(21), 9275–9282. https://doi.org/10.1093/nar/gkr606 Sarita, B., Samadhan, D., Hassan, M. Z., & Kovaleva, E. G. (2025). A comprehensive review of probiotics and human health-current prospective and applications. Frontiers in Microbiology, 15, 1487641. https://doi.org/10.3389/fmicb.2024.1487641 Shah, A. B., Baiseitova, A., Zahoor, M., Ahmad, I., Ikram, M., Bakhsh, A., Shah, M. A., Ali, I., Idress, M., Ullah, R., Nasr, F. A., & Al-Zharani, M. (2024). Probiotic significance of Lactobacillus strains: A comprehensive review on health impacts, research gaps, and future prospects. Gut Microbes, 16(1), 2431643. https://doi.org/10.1080/19490976.2024.2431643 Singh, T. P., Kaur, G., Kapila, S., & Malik, R. K. (2017). Antagonistic Activity of Lactobacillus reuteri Strains on the Adhesion Characteristics of Selected Pathogens. Frontiers in Microbiology, 8, 486. https://doi.org/10.3389/fmicb.2017.00486 van Rooijen, R. J., Gasson, M. J., & de Vos, W. M. (1992). Characterization of the Lactococcus lactis lactose operon promoter: Contribution of flanking sequences and LacR repressor to promoter activity. Journal of Bacteriology, 174(7), 2273–2280. Wang, J., Ji, H., Wang, S., Liu, H., Zhang, W., Zhang, D., & Wang, Y. (2018). Probiotic Lactobacillus plantarum Promotes Intestinal Barrier Function by Strengthening the Epithelium and Modulating Gut Microbiota. Frontiers in Microbiology, 9, 1953. https://doi.org/10.3389/fmicb.2018.01953 Weinberger, A. D., Wolf, Y. I., Lobkovsky, A. E., Gilmore, M. S., & Koonin, E. V. (2012). Viral Diversity Threshold for Adaptive Immunity in Prokaryotes. mBio, 3(6), 10.1128/mbio.00456-12. https://doi.org/10.1128/mbio.00456-12 Westra, E. R., van Houte, S., Gandon, S., & Whitaker, R. (2019). The ecology and evolution of microbial CRISPR-Cas adaptive immune systems. Philosophical Transactions of the Royal Society B: Biological Sciences, 374(1772), 20190101. https://doi.org/10.1098/rstb.2019.0101 Wright, A. V., Sternberg, S. H., Taylor, D. W., Staahl, B. T., Bardales, J. A., Kornfeld, J. E., & Doudna, J. A. (2015). Rational design of a split-Cas9 enzyme complex. Proceedings of the National Academy of Sciences, 112(10), 2984–2989. https://doi.org/10.1073/pnas.1501698112 Wu, Y., Jha, R., Li, A., Liu, H., Zhang, Z., Zhang, C., Zhai, Q., & Zhang, J. (2022). Probiotics (Lactobacillus plantarum HNU082) Supplementation Relieves Ulcerative Colitis by Affecting Intestinal Barrier Functions, Immunity-Related Gene Expression, Gut Microbiota, and Metabolic Pathways in Mice. Microbiology Spectrum, 10(6), e01651-22. https://doi.org/10.1128/spectrum.01651-22 Yu, B., Liu, J. R., Chiou, M. Y., Hsu, Y. R., & Chiou, P. W. S. (2007). The Effects of Probiotic Lactobacillus reuteri Pg4 Strain on Intestinal Characteristics and Performance in Broilers. Asian-Australasian Journal of Animal Sciences, 20(8), 1243–1251. https://doi.org/10.5713/ajas.2007.1243 Yu, B., Liu, J. R., Hsiao, F. S., Lee, T. T., & Chiou, P. W. S. (2008). The Probiotic and Adherence Properties of Lactobacillus reuteri Pg4 Expressing the Rumen Microbial β-Glucanase. Asian-Australasian Journal of Animal Sciences, 21(9), 1324–1329. https://doi.org/10.5713/ajas.2008.70519 尚芊彣(Qian-Wen Shang). (2020, Spring). 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. 國立台灣大學學位論文. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/99133 | - |
| dc.description.abstract | CRISPR-Cas系統是細菌與古菌對抗外來核酸片段的適應性免疫機制。其中，CRISPR-Cas9屬於type II CRISPR-Cas系統，因能有效辨識並準確切割特定DNA序列，目前已廣泛應用於基因編輯技術。Limosilactobacillus reuteri為常見的益生菌，可為宿主提供多項健康益處，相關益生功能之研究甚多，然而對其內源性CRISPR-Cas9系統的探討仍相當有限。本研究針對L. reuteri菌株的內源性CRISPR-Cas9系統進行分析，包括具備此系統菌株的篩選、基因組成、Cas9 蛋白特性及PAM序列辨識偏好等比較。進一步以L. reuteri Pg4菌株為代表，依據PAM預測結果建構用於功能驗證之干擾質體，探討Pg4 Cas9作為基因編輯工具的可行性。分析結果顯示，64株L. reuteri菌株中，僅13株菌株（含Pg4）具備內源性CRISPR-Cas9系統，且不同菌株間的CRISPR array組成與cas基因分布表現出差異。透過Cas9蛋白質多序列比對發現，L. reuteri菌株Cas9蛋白序列具有高度保守性，雖與目前主要用於基因編輯之spCas9存在明顯差異，但在催化殘基與Bridge helix中的重要精胺酸殘基處仍具高度保守性，顯示其潛在的DNA切割能力。此外，AlphaFold預測之三維結構顯示，Pg4 Cas9與其他12株L. reuteri菌株的Cas9結構高度相似，但與spCas9整體結構有顯著差異，特別是在核酸酶葉的HNH結構與PI domain區域，可能影響其DNA切割能力與PAM序列辨識特性。在PAM序列預測方面，首先以Protein2PAM工具，根據Cas9蛋白質序列，預測L. reuteri Cas9 偏好之 PAM 序列皆為5’-NNAAA-3’；進一步針對Pg4菌株，以CRISPRTarget及WebLogo工具分析其 protospacer 序列，預測其偏好辨識的PAM序列為5’-ACAAA-3’，與Protein2PAM預測結果相互呼應。為驗證Pg4 Cas9對PAM序列之辨識效果，本研究已建構含該預測PAM之干擾質體，預期將轉形至Pg4菌株進行功能性驗證。總結而言，本研究透過整合基因體與蛋白質序列分析、三維結構預測、功能驗證實驗設計與干擾質體建構，建立了L. reuteri Pg4內源性CRISPR-Cas9系統之基礎研究框架。不僅拓展對L. reuteri CRISPR-Cas9系統之了解，也為後續深入探討其Cas9蛋白功能與基因編輯應用提供了重要材料與發展方向。 | zh_TW |
| dc.description.abstract | The CRISPR-Cas system is an adaptive immune mechanism in bacteria and archaea that defends against invading nucleic acids. Specifically, the CRISPR-Cas9 system has been widely applied in genome editing due to its efficient and precise DNA cleavage activity. Limosilactobacillus reuteri is a common probiotic known for its health-promoting effects, however, its endogenous CRISPR-Cas9 system remains largely unexplored. In this study, we analyzed the endogenous CRISPR-Cas9 system of L. reuteri strains, including the presence of the system, gene composition, protein characteristics, and PAM sequence preferences. Among 64 L. reuteri strains, only 13 strains, including Pg4, were found to harbor CRISPR-Cas9 systems, exhibiting diversity in CRISPR arrays and cas gene arrangements. Multiple sequence alignment of Cas9 proteins revealed that the Cas9 sequences among L. reuteri strains are highly conserved. Although they show noticeable differences from spCas9, which is commonly used in genome editing, key catalytic residues and critical arginine residues within the bridge helix remain conserved, suggesting potential DNA cleavage activity. AlphaFold-predicted structures revealed that Pg4 Cas9 shares high similarity with other L. reuteri Cas9 proteins but exhibits notable structural differences from spCas9, particularly in the HNH and PI domains, which may affect DNA binding activity or PAM recognition. PAM prediction using Protein2PAM indicated that L. reuteri Cas9 proteins commonly prefer the PAM sequence 5′-NNAAA-3′. Furthermore, CRISPRTarget and WebLogo analyses based on protospacer sequences predicted that the preferred PAM of the Pg4 strain is 5′-ACAAA-3′, consistent with the Protein2PAM result. An interference plasmid containing the predicted PAM was constructed for future validation in the Pg4 strain. In summary, this study establishes a foundational framework for understanding the CRISPR-Cas9 system in L. reuteri Pg4 and provides valuable insights for the future development of strain-specific genome editing tools. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-08-21T16:31:02Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-08-21T16:31:02Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 謝辭 I
中文摘要 II Abstract IV 目 次 VI 圖次 VIII 表次 IX 第一章、序言 1 第二章、文獻探討 2 一、益生菌 2 (一) 定義與認定方式 2 (二) 常見益生菌與益生菌之功能 2 (三) 乳酸桿菌簡介與其應用 3 (四) Lactobacillus reuteri（Limosilactobacillus reuteri） 5 二、CRISPR-Cas系統 9 (一) 系統起源與發展 9 (二) 系統分類、組成與作用機制 10 (三) CRISPR-Cas9系統 11 三、CRISPR-Cas9系統在乳酸桿菌中的應用 15 四、L. reuteri Pg4之研究背景 16 第三章、材料與方法 17 一、研究架構 17 二、基因體序列、Cas9蛋白分析與PAM預測 17 (一) 基因體序列來源 17 (二) CRISPR-Cas9系統辨識 20 (三) L. reuteri菌株Cas9親緣關係樹建構 20 (四) Cas9蛋白質序列與結構比對 21 (五) L. reuteri菌株PAM序列預測 22 三、L. reuteri Pg4菌株PAM序列驗證 23 (一) 使用菌株及來源 23 (二) 培養基 23 (三) 干擾質體建構 23 四、干擾質體轉入L. reuteri Pg4菌株 26 (一) 勝任細胞製備 26 (二) 電穿孔條件 26 五、干擾質體轉形效率 27 第四章、結果 28 一、CRISPR-Cas9系統分析 28 二、Cas9蛋白親緣關係比較與結構分析 32 (一) L. reuteri菌株Cas9蛋白之親緣關係樹 32 (二) Cas9蛋白質序列與結構比對 32 (三) Cas9蛋白質三維結構比對與分析 34 三、L. reuteri菌株PAM序列預測 49 四、Pg4菌株PAM序列驗證之質體建構 54 第五章、討論 58 一、L. reuteri中CRISPR-Cas9系統的多樣性 58 二、Pg4 Cas9的序列特徵、演化關係與結構分析 60 三、PAM序列之預測與干擾質體設計策略 61 四、Pg4 Cas9作為基因編輯工具的潛力與挑戰 62 第六章、結論與未來展望 64 參考文獻 65 附錄 73 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | CRISPR-Cas9 | zh_TW |
| dc.subject | Limosilactobacillus reuteri | zh_TW |
| dc.subject | PAM 序列預測 | zh_TW |
| dc.subject | Cas9 蛋白序列與結構分析 | zh_TW |
| dc.subject | 基因編輯 | zh_TW |
| dc.subject | genome editing | en |
| dc.subject | PAM sequence prediction | en |
| dc.subject | Cas9 protein sequence and structure analysis | en |
| dc.subject | CRISPR-Cas9 | en |
| dc.subject | Limosilactobacillus reuteri | en |
| dc.title | Limosilactobacillus reuteri CRISPR-Cas9系統特性分析 | zh_TW |
| dc.title | Analysis of CRISPR-Cas9 system in Limosilactobacillus reuteri | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 謝建元;彭及忠;劉啟德 | zh_TW |
| dc.contributor.oralexamcommittee | Chien-Yan Hsieh;Chi-Chung Peng;Chi-Te Liu | en |
| dc.subject.keyword | Limosilactobacillus reuteri,CRISPR-Cas9,Cas9 蛋白序列與結構分析,PAM 序列預測,基因編輯, | zh_TW |
| dc.subject.keyword | Limosilactobacillus reuteri,CRISPR-Cas9,Cas9 protein sequence and structure analysis,PAM sequence prediction,genome editing, | en |
| dc.relation.page | 77 | - |
| dc.identifier.doi | 10.6342/NTU202503471 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2025-08-07 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 生物科技研究所 | - |
| dc.date.embargo-lift | 2029-05-01 | - |
| Appears in Collections: | 生物科技研究所 | |
Files in This Item:
| File | Size | Format | |
|---|---|---|---|
| ntu-113-2.pdf Restricted Access | 8.61 MB | Adobe PDF | View/Open |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.