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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52076完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 羅筱鳳(Hsiao-feng Lo) | |
| dc.contributor.author | Syuan-Fei Hong | en |
| dc.contributor.author | 洪瑄斐 | zh_TW |
| dc.date.accessioned | 2021-06-15T14:07:04Z | - |
| dc.date.available | 2017-07-01 | |
| dc.date.copyright | 2015-08-28 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-20 | |
| dc.identifier.citation | Abe, A., S. Kosugi, K. Yoshida, S. Natsume, H. Takagi, H. Kanzaki, H. Matsumura, K. Yoshida, C. Mitsuoka, M. Tamiru, H. Innan, L. Cano, S. Kamoun, and R. Terauchi. 2012. Genome sequencing reveals agronomically important loci in rice using MutMap. Nat. Biotechnol. 30:174-178.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52076 | - |
| dc.description.abstract | 多基因轉殖技術常用於研究植物基因調控與交互作用之關係，須克服轉殖植物基因表現不穩定之障礙。多基因轉植株可經多次轉殖或雜交獲得，但轉殖基因共同分離 (cosegregation)、多拷貝數 (copy number)、或是轉殖基因結構複雜皆可能造成後生遺傳調控現象而干擾轉殖基因表現。因此，我們建立再定序(resequencing) 的分析技術以確認農桿菌轉殖殖株之T-DNA插入點。欲驗證輔助性蛋白酶 (helper component-proteinase, HC-Pro) 利用細胞自噬途徑 (autophagy) 降解 AGRONAUTE1 (AGO1)之假說，以雜交方式將HC-Pro基因導入不同autophagy-related (atgs) 突變株。利用熱不對稱性交錯PCR (Thermal asymmetric interlaced-polymerase chain reaction; TAIL-PCR) 與再定序技術確認P1/HC21-21轉植株的T-DNA插入點，結果顯示P1/HC21-21轉植株基因中含有多個插入片段，且HC-Pro基因與GUS基因鏈鎖。假設中預期atg/P1/HC21-21植物中因細胞自噬缺陷，HC-Pro無法促進AGO1降解，導致AGO1累積且表現較微弱的鋸齒葉葉形。然而，雜交atg突變株與P1/HC21-21轉植株結果顯示，atgs × P1/HC21-21 F2子代可能發生轉錄時基因默化現象 (transcriptional gene silencing)。此外，AGO1與其他相關蛋白之抗體經由親和性層析純化出IgG後，可增加靈敏性與專一性並用於蛋白質分析實驗。了解P1/HC與atg/ P1/HC植物之AGO1表現量可驗證HC-Pro誘導細胞自噬降解AGO1之假說。 | zh_TW |
| dc.description.abstract | In plant science, the major obstacle of the exploration of gene relationship in regulation and interaction is the difficulty to manipulate multiple transgenes. Multiple transgenes can be introduced either by retransformation or by sexual crossing, but transgenic loci cosegregation, copy number, the structure of transgene may cause epigenetic regulation and sometimes interrupt the expression level of transgenes. Here, we established a procedure to identify the T-DNA insertion site in Agrobacterium- mediated transgenic plants by resequencing. The study of helper component-proteinase (HC-Pro), a viral gene silencing suppressor, triggering autophagic AGRONAUTE1 (AGO1) degradation is evaluated through expressing HC-Pro gene in different genetic backgrounds of autophagy-related gene T-DNA insertion mutants (atgs) by crossing. Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and resequencing approaches were used for identification of the T-DNA insertion sites in various transgenic plants. The results indicated that multiple insertions and the linkage between HC-Pro and GUS gene showed in P1/HC21-21 plants. We expected that autophagy-deficiency in atg/P1/HC21-21 plants enhanced AGO1 accumulation and showed milder serrated phenotype. However, normal leaf phenotype in all of the atg8a × P1/HC21-21, atg5 × P1/HC21-21 F2 progenies suggested that transcriptional gene silencing might occur. Besides, antibodies against AGO1, HEN1 and another silencing suppressor p19 were produced with affinity purification for protein assay. Verifying AGO1 expression in P1/HC and atg/ P1/HC plants will help to prove our hypothesis that HC-Pro triggers autophagic AGO1 degradation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T14:07:04Z (GMT). No. of bitstreams: 1 ntu-104-R02628108-1.pdf: 26743259 bytes, checksum: a95ff41dcaaf6f1da19ca6b1304517db (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 論文口試委員審定書 I
謝辭 II 摘要 III Abstract IV Contents VI List of Figures IX List of Supplemental data XI Introduction 1 Identification of T-DNA insertion position in transgenic plant 1 Gene silencing mechanism 2 Viral suppressor triggers AGO1 autophagical degradation 4 Enhancement of antibody sensitivity 5 Materials and methods 8 Plant materials and growth conditions. 8 Plant crossing and genotyping 8 Schematic thermal asymmetric interlaced PCR 9 Resquencing and data analysis 9 Gene construction for recombinant protein production 10 Recombinant protein purification and antiserum production 11 Affinity purification for IgG 11 Immunoblotting 12 Results 14 Confirming T-DNA insertion site of P1/HC 21-21 plants by TAIL-PCR 14 Establishment of resequencing pipeline for T-DNA insertion identification 15 P1/HC21-21 plants resequencing revealed that two insertions are located on chromosome 3 16 P1/HC12-2 plants resequencing showed that the T-DNA is located on chromosome 1 17 Repeated T-DNA insertions were showed in GUS plants line L1 resequencing 18 Specific primers designed for P1/HC21-21 plants genotyping 19 Specific primers designed for P1/HC12-2 plants genotyping 20 Specific primers designed for GUS plants line L1 genotyping 21 Loss of serrated-leaf phenotype after crossing P1/HC 21-21 plants and atg mutants 21 AGO1 antiserum production and evaluation 23 Improvement of AGO1 IgG efficiency with affinity Purification 24 P19 antibody production 25 NtHEN1a antibody production 25 Discussions 27 Identification of T-DNA insertion in transgenic plants 27 The complexity of Agrobacterium-mediated T-DNA insertion 28 Identification of T-DNA insertion on GUS plants line L1 29 Gene silencing occurred in atgs and P1/HC21-21 plants crossing 29 The benefit of antibody production by affinity purification in FPLC system 32 References 35 Figures 46 Supplemental data 62 | |
| dc.language.iso | en | |
| dc.subject | 農桿菌轉殖技術 | zh_TW |
| dc.subject | 細胞自噬 | zh_TW |
| dc.subject | 助性蛋白? | zh_TW |
| dc.subject | 病毒抑制子 | zh_TW |
| dc.subject | 轉錄時基因默化現象 | zh_TW |
| dc.subject | 再定序 | zh_TW |
| dc.subject | 抗原親和性管住層析 | zh_TW |
| dc.subject | SALK突變株 | zh_TW |
| dc.subject | T-DNA插入點 | zh_TW |
| dc.subject | 熱不對稱性交錯PCR | zh_TW |
| dc.subject | T-DNA identification | en |
| dc.subject | antigen-specific affinity chromatography | en |
| dc.subject | transcriptional gene silencing | en |
| dc.subject | resequencing | en |
| dc.subject | viral suppressor | en |
| dc.subject | helper component-proteinase | en |
| dc.subject | autophagy | en |
| dc.subject | Agrobacterium-mediated transformation | en |
| dc.subject | thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) | en |
| dc.subject | SALK lines | en |
| dc.title | 建立T-DNA插入點分析與親和性抗體純化技術探討HC-Pro誘導細胞自噬途徑降解AGO1之機制 | zh_TW |
| dc.title | Establishment of T-DNA Insertion Identification and Antibodies Affinity Purification for Studying the Mechanism on Autophagic AGO1 Degradation by HC-Pro | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 林詩舜(Shih-Shun Lin) | |
| dc.contributor.oralexamcommittee | 林劭品(Sau-Ping Lin),林淑怡(Shu-I Lin) | |
| dc.subject.keyword | 病毒抑制子,助性蛋白?,細胞自噬,農桿菌轉殖技術,熱不對稱性交錯PCR,T-DNA插入點,SALK突變株,再定序,轉錄時基因默化現象,抗原親和性管住層析, | zh_TW |
| dc.subject.keyword | viral suppressor,helper component-proteinase,autophagy,Agrobacterium-mediated transformation,thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR),T-DNA identification,SALK lines,resequencing,transcriptional gene silencing,antigen-specific affinity chromatography, | en |
| dc.relation.page | 75 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-08-20 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 園藝暨景觀學系 | zh_TW |
| 顯示於系所單位： | 園藝暨景觀學系 | |
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