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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李昆達 | zh_TW |
| dc.contributor.advisor | Kung-Ta Lee | en |
| dc.contributor.author | 蔣尚伶 | zh_TW |
| dc.contributor.author | Shan-Ling Chiang | en |
| dc.date.accessioned | 2023-07-11T16:06:25Z | - |
| dc.date.available | 2023-07-12 | - |
| dc.date.copyright | 2023-07-11 | - |
| dc.date.issued | 2022 | - |
| dc.date.submitted | 2002-01-01 | - |
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R., & Medina, M. I. (2009). In situ and de novo biosynthesis of vitamin C in wild type and transgenic tomato hairy roots: A precursor feeding study. Plant Science, 177(1), 28-34. Yin, D., Liu, W., Zhang, Y., & Sun, X. (2021). Differential expression of pinoresinol-lariciresinol reductase gene in relation to podophyllotoxin accumulation in different plant organs of endangered anticancer species Podophyllum peltatum. Pakistan Journal of Botany, 53(4). Yousefian, S., Lohrasebi, T., Farhadpour, M., & Haghbeen, K. (2020). Effect of methyl jasmonate on phenolic acids accumulation and the expression profile of their biosynthesis-related genes in Mentha spicata hairy root cultures. Plant cell, tissue and organ culture, 142(2), 285-297. Zupan, J., Muth, T. R., Draper, O., & Zambryski, P. (2000). The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. The Plant Journal, 23(1), 11-28. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/87647 | - |
| dc.description.abstract | 鬼臼素是由鬼臼屬植物中分離出具有抗病毒能力的一種木脂素，其衍生物etoposide為可以抑制癌細胞的有絲分裂而做為化療藥物使用。鬼臼素在天然資源中相當少見，因此很難滿足對其日益增長的需求，因此，如何大量生產鬼臼毒素及其衍生物的新方法是一個迫在眉睫的問題。羅勒是一種富含酚酸和萜類化合物的草本植物，迷迭香酸、咖啡酸和綠原酸是羅勒中含量最豐富的酚類化合物，在先前的研究中曾進行迷迭香酸合成酶的RNA干擾（RASi）以增加苯基類丙烷化合物前驅物的累積，鬼臼素生合成的第一步是透過松脂醇-落葉松樹脂醇還原酶 （PLR）將松脂醇轉化為secoisolariciresinol。本研究於煙草葉片中進行八角蓮PLR瞬時表達，以了解其在植物細胞中的表現位置及活性。 PLR可同時在表皮細胞的細胞質和細胞核中被觀察到。在體外測試中則未檢測到PLR的催化活性。接著，將PLR轉形或與RASi一起轉形進羅勒毛狀根以生產開環異落葉松樹脂醇。在這些毛狀根的代謝物分析中，僅在PLR與RASi毛狀根中發現濃度為0.008 ppm/毫克乾重的松脂醇，在其它毛狀根中未觀察到PLR的其他產物落葉松樹脂醇和secoisolariciresinol。接下來將測量羅勒毛狀根中PLR的RNA和蛋白質表現量，並進一步分析其的代謝體學，以強化PLR的生產效率。 | zh_TW |
| dc.description.abstract | Podophyllotoxin is a lignan isolated from Podophyllum species and has antivirus ability. The derivative, etoposide, is also used as a medication for chemotherapy to inhibit cancer cell mitosis. Podophyllotoxin is rare in the natural source so it is hard to satisfy the growing demand. A new strategy to produce podophyllotoxin and its derivatives is an urgent problem. Ocimum basilicum (basil) is a valuable herb that is rich in phenolic acids and terpenoids. Rosmarinic acid, caffeic acid, and chlorogenic acid are the most abundant phenolic compounds in basil. The RNA interference of rosmarinic acid synthase (RASi) was expressed in the previous study to enrich the phenylpropanoids precursor accumulation. The first step of podophyllotoxin biosynthesis is to convert pinoresinol into secoisolariciresinol by the enzyme pinoresinol-lariciresinol reductase (PLR). In this study, PLR from Podophyllum pleianthum transient expression in the leaves of Nicotiana benthamiana was conducted first to realize its subcellular localization and activity in plants. PLR was expressed in the cytoplasm and nucleus of epidermal cells simultaneously. However, the catalytic activity of PLR was not detected in the in vitro test. Then, PLR was transformed into the basil hairy roots individually or with the RASi construction to the synthesis of secoisolariciresinol. In the metabolic analysis of these hairy roots, only pinoresinol was found in the PLR with RASi hairy roots with a low concentration of 0.008 ppm/mg DW. Other products of PLR were not observed in the hairy roots. The RNA level and protein expression of PLR will be determined, and the metabolome of basil hairy root will be further analyzed in the next step to optimize PLR expression in hairy roots for podophyllotoxin biosynthesis. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-07-11T16:06:25Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-07-11T16:06:25Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 誌謝 i
Abstract ii 中文摘要 iii Abbreviation iv Content vi List of Tables and Figures ix List of Appendixes x Chapter 1: Introduction 1 1.1 Ocimum basilicum (basil) 1 1.1.1 Ocimum basilicum 1 1.1.2 Metabolites and biosynthesis in Ocimum basilicum 1 1.2 Agrobacterium 2 1.2.1 Agrobacterium rhizogenes 2 1.2.2 Agrobacterium tumefaciens 2 1.3 Hairy roots 3 1.3.1 Hairy roots feature 3 1.3.2 Ocimum basilicum hairy roots 4 1.3.3 Rosmarinic acid synthase RNA interference (RASi) 5 1.4 Podophyllotoxin 5 1.4.1 Podophyllotoxin biosynthesis 5 1.4.2 Pinoresinol-lariciresinol reductase (PLR) 6 Chapter 2: Objective 9 Chapter 3: Materials and methods 10 3.1 Plant materials 10 3.1.1 Ocimum basilicum culture 10 3.1.2 Induction of Ocimum basilicum hairy roots 10 3.1.3 Confirmation of Ocimum basilicum hairy roots 10 3.1.4 Nicotiana benthamiana culture 11 3.2 DNA preparation 11 3.2.1 Plasmid extraction 11 3.2.2 DNA agarose gel electrophoresis 12 3.2.3 DNA purification 12 3.2.4 DNA quantification 12 3.2.5 Polymerase chain reaction (PCR) 12 3.3 Plasmid construction 13 3.3.1 Construction of PLR transient expression vector 13 3.3.2 Construction of PLR expression vector 13 3.3.3 Construction of PLR and RASi co-expression vector 14 3.3.4 Escherichia coli transformation 14 3.3.5 Agrobacterium competent cells preparation 14 3.3.6 Agrobacterium tumefaciens and Agrobacterium rhizogenes transformation 14 3.4 Transient expression 15 3.5 Enzyme activity analysis in vitro 15 3.6 Metabolites analysis 16 3.6.1 Metabolites extraction 16 3.6.2 HPLC analysis 16 3.6.3 LC-MS/MS analysis 16 Chapter 4: Results 17 4.1 PLR transient expression in Nicotiana benthamiana 17 4.1.1 Transient expression vector construction 17 4.1.2 PLR subcellular localization analysis 17 4.1.3 PLR enzyme activity analysis in vitro 17 4.2 PLR and PLR with RASi Ocimum basilicum hairy roots metabolites analysis 18 4.2.1 PLR and PLR with RASi expression vector construction 18 4.2.2 PLR and PLR with RASi Ocimum basilicum hairy roots induction 19 4.2.3 Ocimum basilicum hairy roots metabolites analysis by LC-MS/MS 19 Chapter 5: Discussion 23 5.1 PLR transient expression in Nicotiana benthamiana 23 5.1.1 PLRs from other species express in the cytosol 23 5.1.2 PpPLR expresses in the cytosol and nucleus 23 5.1.3 PLR did not present its catalytic activity in the leaves of N. benthamiana in vitro activity analysis 25 5.2 Metabolites analysis of the transgenic hairy roots 26 5.2.1 Lariciresinol m/z is not correspond to the prediction 26 5.2.2 The effect of RASi construction in the basil hairy roots 26 5.2.3 No product is catalyzed by PLR in the PLR with RASi combination hairy roots 27 5.3 Future work 28 Chapter 6: Conclusion 30 Tables 31 Figures 35 Appendixes 50 Reference 67 | - |
| dc.language.iso | en | - |
| dc.title | 八角蓮的松脂醇-落葉松樹脂醇還原酶於菸草葉片與羅勒毛狀根表現之研究 | zh_TW |
| dc.title | Expression of pinoresinol-lariciresinol reductase from Podophyllum pleianthum in Nicotiana benthamiana leaves and Ocimum basilicum hairy roots | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 110-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 賴爾珉 | zh_TW |
| dc.contributor.oralexamcommittee | Chien-Chih Yang;Nai-Chun Lin;Hieng-Ming Ting;Erh-Min Lai | en |
| dc.subject.keyword | 羅勒,毛狀根,松脂醇-落葉松樹脂醇還原酶,松脂醇,代謝工程, | zh_TW |
| dc.subject.keyword | Ocimum basilimum,hairy roots,pinoresinol-lariciresinol reductase,pinoresinol,metabolic engineering, | en |
| dc.relation.page | 73 | - |
| dc.identifier.doi | 10.6342/NTU202202405 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2022-08-16 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科技學系 | - |
| dc.date.embargo-lift | 2027-08-01 | - |
| 顯示於系所單位： | 生化科技學系 | |
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