請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90194
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林詩舜 | zh_TW |
dc.contributor.advisor | Shih-Shun Lin | en |
dc.contributor.author | 楊芊燕 | zh_TW |
dc.contributor.author | Qian Yuan Yong | en |
dc.date.accessioned | 2023-09-22T17:48:13Z | - |
dc.date.available | 2023-11-09 | - |
dc.date.copyright | 2023-09-22 | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2023-08-09 | - |
dc.identifier.citation | Anandalakshmi, R., Pruss, G.J., Ge, X., Marathe, R., Mallory, A.C., Smith, T.H., and Vance, V.B. (1998). A viral suppressor of gene silencing in plants. Proc Natl Acad Sci U S A 95, 13079-13084.
Anders Hafrén, S.Ü., Anton Hochmuth, Steingrim Svenning, Terje Johansen, Daniel Hofius (2018). Turnip Mosaic Virus Counteracts Selective Autophagy of the Viral Silencing Suppressor HCpro. Barman, A., Deb, B., and Chakraborty, S. (2020). A glance at genome editing with CRISPR-Cas9 technology. Curr Genet 66, 447-462. Berruezo, F., de Souza, F.S.J., Picca, P.I., Nemirovsky, S.I., Martinez-Tosar, L., Rivero, M., Mentaberry, A.N., and Zelada, A.M. (2016). Sequencing of small RNAs of the fern Pleopeltis minima (Polypodiaceae) offers insight into the evolution of the microRNA repertoire in land plants. Bowman, J.L., Kohchi, T., Yamato, K.T., Jenkins, J., Shu, S., Ishizaki, K., Yamaoka, S., Nishihama, R., Nakamura, Y., Berger, F., Adam, C., Aki, S.S., Althoff, F., Araki, T., Arteaga-Vazquez, M.A., Balasubrmanian, S., Barry, K., Bauer, D., Boehm, C.R., Briginshaw, L., Caballero-Perez, J., Catarino, B., Chen, F., Chiyoda, S., Chovatia, M., Davies, K.M., Delmans, M., Demura, T., Dierschke, T., Dolan, L., Dorantes-Acosta, A.E., Eklund, D.M., Florent, S.N., Flores-Sandoval, E., Fujiyama, A., Fukuzawa, H., Galik, B., Grimanelli, D., Grimwood, J., Grossniklaus, U., Hamada, T., Haseloff, J., Hetherington, A.J., Higo, A., Hirakawa, Y., Hundley, H.N., Ikeda, Y., Inoue, K., Inoue, S.I., Ishida, S., Jia, Q., Kakita, M., Kanazawa, T., Kawai, Y., Kawashima, T., Kennedy, M., Kinose, K., Kinoshita, T., Kohara, Y., Koide, E., Komatsu, K., Kopischke, S., Kubo, M., Kyozuka, J., Lagercrantz, U., Lin, S.S., Lindquist, E., Lipzen, A.M., Lu, C.W., De Luna, E., Martienssen, R.A., Minamino, N., Mizutani, M., Mizutani, M., Mochizuki, N., Monte, I., Mosher, R., Nagasaki, H., Nakagami, H., Naramoto, S., Nishitani, K., Ohtani, M., Okamoto, T., Okumura, M., Phillips, J., Pollak, B., Reinders, A., Rovekamp, M., Sano, R., Sawa, S., Schmid, M.W., Shirakawa, M., Solano, R., Spunde, A., Suetsugu, N., Sugano, S., Sugiyama, A., Sun, R., Suzuki, Y., Takenaka, M., Takezawa, D., Tomogane, H., Tsuzuki, M., Ueda, T., Umeda, M., Ward, J.M., Watanabe, Y., Yazaki, K., Yokoyama, R., Yoshitake, Y., Yotsui, I., Zachgo, S., and Schmutz, J. (2017). Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome. Cell 171, 287-304 e215. Bu, F., Yang, M., Guo, X., Huang, W., and Chen, L. (2020). Multiple Functions of ATG8 Family Proteins in Plant Autophagy. Front Cell Dev Biol 8, 466. Derkacheva, M., Steinbach, Y., Wildhaber, T., Mozgova, I., Mahrez, W., Nanni, P., Bischof, S., Gruissem, W., and Hennig, L. (2013). Arabidopsis MSI1 connects LHP1 to PRC2 complexes. EMBO J 32, 2073-2085. Dong, Z., Han, M.H., and Fedoroff, N. (2008). The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc Natl Acad Sci U S A 105, 9970-9975. Feng, J., and Lu, J. (2017). LHP1 Could Act as an Activator and a Repressor of Transcription in Plants. Front Plant Sci 8, 2041. Frei dit Frey, N., Muller, P., Jammes, F., Kizis, D., Leung, J., Perrot-Rechenmann, C., and Bianchi, M.W. (2010). The RNA binding protein Tudor-SN is essential for stress tolerance and stabilizes levels of stress-responsive mRNAs encoding secreted proteins in Arabidopsis. Plant Cell 22, 1575-1591. Fukudome, A., and Fukuhara, T. (2017). Plant dicer-like proteins: double-stranded RNA-cleaving enzymes for small RNA biogenesis. J Plant Res 130, 33-44. Gutierrez-Beltran, E., Moschou, P.N., Smertenko, A.P., and Bozhkov, P.V. (2015). Tudor staphylococcal nuclease links formation of stress granules and processing bodies with mRNA catabolism in Arabidopsis. Plant Cell 27, 926-943. Gutierrez-Beltran, E., Denisenko, T.V., Zhivotovsky, B., and Bozhkov, P.V. (2016). Tudor staphylococcal nuclease: biochemistry and functions. Cell Death Differ 23, 1739-1748. Han, M.H., Goud, S., Song, L., and Fedoroff, N. (2004). The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc Natl Acad Sci U S A 101, 1093-1098. Hong, S.F. (2023). The functional investigation of RNA-induced silencing complex in Marchantia polymorpha and Arabidopsis. Hong, S.F., Fang, R.Y., Wei, W.L., Jirawitchalert, S., Pan, Z.J., Hung, Y.L., Pham, T.H., Chiu, Y.H., Shen, T.L., Huang, C.K., and Lin, S.S. (2023). Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-Pro. Virol J 20, 10. Hu, S.F., Wei, W.L., Hong, S.F., Fang, R.Y., Wu, H.Y., Lin, P.C., Sanobar, N., Wang, H.P., Sulistio, M., Wu, C.T., Lo, H.F., and Lin, S.S. (2020). Investigation of the effects of P1 on HC-pro-mediated gene silencing suppression through genetics and omics approaches. Bot Stud 61, 22. Ishizaki, K., Chiyoda, S., Yamato, K.T., and Kohchi, T. (2008). Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L., an emerging model for plant biology. Plant Cell Physiol 49, 1084-1091. Kasschau, K.D., Xie, Z., Allen, E., Llave, C., Chapman, E.J., Krizan, K.A., and Carrington, J.C. (2003). P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction. Dev Cell 4, 205-217. Kim, G.T., Tsukaya, H., and Uchimiya, H. (1998). The CURLY LEAF gene controls both division and elongation of cells during the expansion of the leaf blade in Arabidopsis thaliana. Planta 206, 175-183. Kubota, A., Ishizaki, K., Hosaka, M., and Kohchi, T. (2014). EfficientAgrobacterium-Mediated Transformation of the LiverwortMarchantia polymorphaUsing Regenerating Thalli. Bioscience, Biotechnology, and Biochemistry 77, 167-172. Lin, P.C., Lu, C.W., Shen, B.N., Lee, G.Z., Bowman, J.L., Arteaga-Vazquez, M.A., Liu, L.Y., Hong, S.F., Lo, C.F., Su, G.M., Kohchi, T., Ishizaki, K., Zachgo, S., Althoff, F., Takenaka, M., Yamato, K.T., and Lin, S.S. (2016). Identification of miRNAs and Their Targets in the Liverwort Marchantia polymorpha by Integrating RNA-Seq and Degradome Analyses. Plant Cell Physiol 57, 339-358. Lin, S.S., and Bowman, J.L. (2018). MicroRNAs in Marchantia polymorpha. New Phytol 220, 409-416. Liu, J., Deng, S., Wang, H., Ye, J., Wu, H.W., Sun, H.X., and Chua, N.H. (2016). CURLY LEAF Regulates Gene Sets Coordinating Seed Size and Lipid Biosynthesis. Plant Physiol 171, 424-436. Lu, C., and Fedoroff, N. (2000). A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin. Plant Cell 12, 2351-2366. Parent, J.S., Bouteiller, N., Elmayan, T., and Vaucheret, H. (2015). Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing. Plant J 81, 223-232. Re, D.A., Cambiagno, D.A., Arce, A.L., Tomassi, A.H., Giustozzi, M., Casati, P., Ariel, F.D., and Manavella, P.A. (2020). CURLY LEAF Regulates MicroRNA Activity by Controlling ARGONAUTE 1 Degradation in Plants. Mol Plant 13, 72-87. Singkaravanit-Ogawa, S., Kosaka, A., Kitakura, S., Uchida, K., Nishiuchi, T., Ono, E., Fukunaga, S., and Takano, Y. (2021). Arabidopsis CURLY LEAF functions in leaf immunity against fungal pathogens by concomitantly repressing SEPALLATA3 and activating ORA59. Plant J 108, 1005-1019. Sugano, S.S., Nishihama, R., Shirakawa, M., Takagi, J., Matsuda, Y., Ishida, S., Shimada, T., Hara-Nishimura, I., Osakabe, K., and Kohchi, T. (2018). Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha. PLoS One 13, e0205117. Tjita, V. (2021). Investigation of miRNA biogenesis pathway related genes in Marchantia polymorpha. Vazquez, F., Gasciolli, V., Crete, P., and Vaucheret, H. (2004). The nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing. Curr Biol 14, 346-351. Wei, W.-L., Tran, P.-A., Fang, R.-Y., Pham, T.H., Bowman, J., Hong, S.-F., Pan, Z.-J., Shang, Q.-W., Lin, P.-C., Shen, B.-N., Wu, F.-H., Lin, C.-S., Shen, T.-L., and Lin, S.-S. (2022). The additional unique ability of TuMV HC-Pro in inhibiting HEN1 activity for enhancing the autophagic AGO1 degradation in RNA silencing suppression. Research Square. Wieczorek, P., Jarmolowski, A., Szweykowska-Kulinska, Z., Kozak, M., and Taube, M. (2023). Solution structure and behaviour of the Arabidopsis thaliana HYL1 protein. Biochim Biophys Acta Gen Subj 1867, 130376. Ying, A.C.Z. (2022). Investigation of post-transcriptional gene silencing pathway related genes in Arabidopsis thaliana. | - |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90194 | - |
dc.description.abstract | 基因沉默是個植物中為抵禦微生物或面對環境改變時常見的處變機制。Dicer-like 4 (DCL4) 是植物體中負責切割核糖核酸並產生可與Argonaute 1蛋白 (AGO1)結合之短干擾核糖核酸 (short interfering RNA, siRNA),反式作用干擾小核糖核酸 (trans-acting siRNA),或病毒小核糖核酸(viral small RNA)。Hyponastic Leaves 1 (HYL1) 蛋白是個雙股核糖核酸連接蛋白,是小分子核糖核酸(microRNA, miRNA)生產路徑中一個重要的調節蛋白。AGO1為核糖核酸誘導沉默複合物(RNA-induced silencing complex, RISC)的重要組合物之一,已被證明在地錢中會被MIR11707所調控。常間回文重複序列叢集關聯蛋白(Clustered Regularly Interspaced Short Palindromic Repeat/ CRISPR associated protein 9, CRISPR-Cas9)基因編輯技術在此研究中被應用於變異地錢及阿拉伯介中相關的調節者。另外,葡萄球菌核酸酶样tudor结构域蛋白1 (Tudor-SN, TSN),捲曲葉蛋白(Curly leaf, CLF),和類異染色質蛋白1 (Like Heterochromatin Protein 1, LHP1)皆為植物調節機制中的重要調節者,TSN與信使核糖核酸(messenger RNA, mRNA)的去帽路徑相關,CLF和LHP1則與阿拉伯介的開花相關。因此,我們假設這些蛋白質可以它們的專有方式影響AGO1的功能。協同性蛋白水解酶 (helper-component proteinase; HC-Pro) 是個來自馬鈴薯Y病毒屬的RNA沉默抑制蛋白,它可以影響被感染的植物體内核糖核酸誘導沉默複合物的活動。爲了瞭解HC-Pro和AGO1之間的聯係,我們的前輩把P1/HC-ProTu 轉植株中的自噬蛋白ATG8a去除。在研究的下一步,我們把這個轉植株進行了與Col-0的雜交,由此間接把ATG8a恢復。此轉植株預計可以恢復ATG8a的AGO1降解的功能。除此之外,我們也成功製造出TSN,CLF和LHP1基因的變異植株。在此論文中,我們利用體外核糖核酸誘導沉默複合物活動測定(in vitro RISC activity assay)研究了AGO1蛋白質在各個轉植株及變異植株中的活動。作爲地錢中的模範植物,Marchantia polymorpha在近期植物學研究一直是非常重要的研究模本。利用CRISPR-Cas9的技術,通過把向導RNA (guide RNA, gRNA)片段插入載體並轉入地錢中,我們將DCL4, HYL1和MIR11707由Tak-1中變異去除。我們將會觀察各變異植株的表性特徵。 | zh_TW |
dc.description.abstract | Gene silencing is a typical pathway in plants for defending from viruses and environmental changes. Dicer-like 4 (DCL4) is a dicer protein in plant responses to produce short-interfering RNA (siRNA) for trans-acting siRNA (tasiRNA) and viral small RNA that could be loaded into Argonaute 1 (AGO1) for gene silencing, whereas Hyponastic Leaves 1 (HYL1) is a double strand RNA binding protein that is one of the regulators in the microRNA (miRNA) biogenesis pathway. As an essential component in the RNA-induced silencing complex (RISC), AGO1 is also known to be regulated by miR11707 in Marchantia polymorpha. To further investigate the roles of these regulators, CRISPR-Cas9 gene editing has been used to generate knocked-out mutant in Arabidopsis and M. polymorpha plants in this study. Moreover, Tudor-SN (TSN), Curly Leaf (CLF), and Like Heterochromatin Protein1 (LHP1) proteins were shown to be important in plant regulatory pathways, whereas TSN related to uncapping of mRNA, CLF, and LHP1 are both related to the flowering of Arabidopsis thaliana. So, we hypothesized that they affect AGO1 function in their specific ways. Helper component-proteinase (HC-Pro) is a viral RNA silencing suppressor from potyvirus that will disrupt the RISC activity in the virus-infected plant. To study the relation between HC-Pro and AGO1, our senior produced a P1/HC-Pro transgenic line that ATG8a is being knocked out simultaneously. As the study's next step, they cross this transgenic line with Columbia (Col-0) to recover the atg8a mutant activity. It is expected that, in this transgenic line, the degradation of AGO1 shall be recovered compared to the atg8a knocked-out line. We had also successfully mutated TSN, CLF, and LHP1 genes in A. thaliana Col-0 ecotype. In this thesis, we studied AGO1 protein levels and in vivo RISC activity assay in all transgenic lines to confirm the AGO1 activity in those lines. As a model plant in liverwort, M. polymorpha has been a critical study model in the plant-related study. With CRISPR-Cas-9, we have knocked out DCL4, HYL1, and MIR11707 genes by inserting a guide RNA (gRNA) fragment in a vector and transferring the vectors into M. polymorpha plants. We will be studying their phenotypic symptoms when these regulators are knocked-out successfully. | en |
dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-09-22T17:48:13Z No. of bitstreams: 0 | en |
dc.description.provenance | Made available in DSpace on 2023-09-22T17:48:13Z (GMT). No. of bitstreams: 0 | en |
dc.description.tableofcontents | Acknowledgement I
中文摘要 II Abstract IV Introduction 1 Materials and methods 6 Plant materials and growth conditions 6 Vector construction and CRISPR/Cas-9-mediated mutations 6 Gene editing for Arabidopsis with CRISPR-Cas9 7 Genotyping 7 Western blot analysis 8 AGO1-IP and in vitro RISC assay 8 Result 11 CRISPR-Cas9 mediated M. polymorpha HYL1 mutation 11 CRISPR-Cas9 mediated M. polymorpha DCL4 mutation 12 CRISPR-Cas9 mediated M. polymorpha MIR11707 mutation 13 AGO1 studies on P1/HC-ProTu transgenic lines. 15 AGO1 studies on tsn1tsn2ge transgenic lines. 17 AGO1 studies on clfge transgenic lines. 18 AGO1 studies on lhp1ge transgenic lines. 20 Discussion 22 Mutation of DCL4, HYL1, and MIR11707 in M. polymorpha by thallus transformation produced a more stable phenotype on CRISPR-Cas9 generated mutants. 22 Unexpected variation between P1/HC-ProTu and P1/HC-ProTu/ATG8a +/+ transgenic lines could be resulted by unforeseen epigenetic changes. 24 Further studies on interaction of TSN1 and TSN2 with P1 and AGO1. 25 Further studies on LHP1 and CLF in AGO1 activity. 25 In vitro RISC activity assay is a good technique to study AGO1 activity in different mutants. 26 Conclusion 27 References 28 Tables 34 Table 1. Summary on all constructs involved in this study. 34 Table 2. guide RNA (gRNA) sequences for CRISPR-Cas9 editing. 35 Table 3. Primers used in this study. 37 Figures 40 Figure 1. CRISPR-Cas9-mediated mutation on MpHYL1. 40 Figure 2. Sequence alignment and protein detection of Mphyl1ge mutants 41 Figure 3. Phenotypic observation of Mphyl1ge mutants. 42 Figure 4. Temperature dependence phenotypic observation of Mphyl1ge plant 43 Figure 5. CRISPR-Cas9-mediated mutation on MpDCL4. 44 Figure 6. Phenotypic observation of Mpdcl4ge mutants 45 Figure 7. CRISPR-Cas9-mediated mutation on MpMIR11707 46 Figure 8. Phenotypic study on Mpmir11707ge mutant 47 Figure 9. Morphological study of P1/HC-ProTu/atg8a +/+ transgenic lines 48 Figure 10. AGO1 studies on Arabidopsis thaliana P1/HC-ProTu/atg8a +/+ transgenic plant. 49 Figure 11. Morphological studies on Arabidopsis thaliana tsn1tsn2ge transgenic plant. 50 Figure 12. Sequencing result on tsn1tsn2ge plant. 51 Figure 13. AGO1 studies on tsn1tsn2ge mutant line. 52 Figure 14. Morphological studies on Arabidopsis thaliana clfge transgenic plant. 53 Figure 15. Sequencing result on clfge mutant. 54 Figure 16. AGO1 activity studies on clfge mutant line. 55 Figure 17. Morphological studies on Arabidopsis thaliana lhp1ge transgenic plant. 56 Figure 18. AGO1 activity studies on lhp1ge mutant line. 57 | - |
dc.language.iso | en | - |
dc.title | 利用基因編輯與體外RISC分析研究蘚苔植物與被子植物微型核酸調控機制 | zh_TW |
dc.title | The investigation for miRNA machinery in bryophyte and angiosperm through CRISPR gene editing and in vitro RISC assay | en |
dc.type | Thesis | - |
dc.date.schoolyear | 111-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 陳荷明;邱子珍;楊俊逸 | zh_TW |
dc.contributor.oralexamcommittee | Ho-Ming Chen;Tzyy-Jen Chiou;Jun-Yi Yang | en |
dc.subject.keyword | 小分子核糖核酸,地錢,協同性蛋白水解酶,基因沉默機制,阿拉伯介, | zh_TW |
dc.subject.keyword | miRNA,Marchantia polymorpha,HC-Pro,gene silencing,Arabidopsis thaliana, | en |
dc.relation.page | 57 | - |
dc.identifier.doi | 10.6342/NTU202303945 | - |
dc.rights.note | 同意授權(全球公開) | - |
dc.date.accepted | 2023-08-12 | - |
dc.contributor.author-college | 生物資源暨農學院 | - |
dc.contributor.author-dept | 生物科技研究所 | - |
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 | |
---|---|---|---|
ntu-111-2.pdf | 5.95 MB | Adobe PDF | 檢視/開啟 |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。