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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭石通 | |
dc.contributor.author | Hsin-Mei Wang | en |
dc.contributor.author | 王幸美 | zh_TW |
dc.date.accessioned | 2021-06-08T02:08:43Z | - |
dc.date.copyright | 2016-02-24 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-01-29 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19615 | - |
dc.description.abstract | Three major classes of plant pigments are flavonoids/anthocyanins, carotenoids and betalains. In this thesis, the effects of the genes encoding flavonoids and carotenoids on metabolites, and the phenotypic changes in transformants by Agrobacterium-mediated transformation were studied.
Agrobacterium-mediated transformation and genetic assay of chalcone synthase in the medicinal plant Echinacea pallida. Echinacea pallida is widely used as an herbal medicine, and is a subject of research in Europe and North America. Micropropagation of Echinacea pallida has been established, but the efficiency of the rooting was low. However, the efficiency of lateral root initiation was increased and the reduction of callus hyperhydricity was improved by supplying gibberellins (GA) instead of auxin. Agrobacterium-mediated transformation was established in Echinacea pallida by modification of the method previously used for E. purpurea (Wang and To, 2004). The most important factor for the transformation of E. pallida was the introduction of a pre-selection phase in which E. pallida leaves were sensitive to antibiotics. The chalcone synthase (CHS) is a key enzyme in the biosynthesis of diverse flavonoids. Here, we investigated the functions of two clusters CHSs, EpaCHS-A and EpaCHS-B, which includes EpaCHS-B1and EpaCHS-B2, isolated from E. pallida to analyze their roles and gene expression in the Petunia CHS transgenic plants. EpaCHS-A and EpaCHS-B1 were found abundantly in petals, whereas EpaCHS-B2 expression was mainly detected in leaves. Nevertheless, the expression of EpaCHSs remained constant in leaves and roots of transgenic E. pallida overexpressing Petunia CHS, while EpaCHS-B2 expression changed in flowers of transgenic plants. In addition, overexpression of Petunia CHS also affected the secondary metabolites in leaves and roots of E. pallida. Modification of flower color by suppressing β-ring carotene hydroxylase genes in Oncidium. Oncidium Gower Ramsey (Onc. Gower Ramsey) is a popular cut flower, but its color is limited to bright yellow. The β-ring carotene hydroxylase (BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH-A2, BCH-B2, and BCH-C2, isolated from Onc. GR to analyze their roles in flower color. RT-PCR expression profiling suggested that BCH2 genes were mainly expressed in flowers. The expression of BCH-B2 remained constant while that of BCH-A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference (RNAi), we created transgenic Oncidium plants with downregulated BCH expression. In the transgenic plants, flower color changed from the bright yellow of the wild-type to light- and white-yellow. BCH-A2 and BCH-B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow coloration. HPLC analysis of the carotenoid composition of the transgenic flowers suggested major reduction in neoxanthin and violaxanthin pigments. Conclusively, BCH2 expression regulated the accumulation of yellow pigments in Oncidium flowers, and the downregulation of BCH-A2 and BCH-B2 changed the flower color from bright yellow to light- and white-yellow. Functional studies of pigmentation genes in E. pallida and Oncidium by transformation and metabolite analysis show that overexpression of Petunia CHS gene in E. pallida or the repression BCH2 genes in transgenic Oncidium profoundly affects the metabolites of pigment biosynthesis in both transformants. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T02:08:43Z (GMT). No. of bitstreams: 1 ntu-105-D96b42004-1.pdf: 16810649 bytes, checksum: a607a66fbae4feb7953d8f724b131473 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | Acknowledgements....................................................i
摘要...........................................................ii Table of contents....................................................1 Abstract............................................................4 Tables.........................................................7 Figures........................................................8 Abbreviations.......................................................10 1. Chapter 1: In vitro regeneration, Agrobacterium-mediated transformation and genetic assay of chalcone synthase in the medicinal plant Echinacea pallida. .........................................................11 Abstract............................................................11 Introduction.........................................................12 Materials and Methods................................................17 (1) Plant materials...................................................17 (2) Plant regeneration from in vitro leaf explants...........................17 (3) Plasmid construction..............................................18 (4) Genetic transformation of E. pallida..................................18 (5) DNA isolation, polymerase chain reaction (PCR) and Southern blotting......19 (6) DIG probe..................................................19 (7) RNA extraction, RT-PCR and real-time PCR...........................20 (8) Rapid amplification of cDNA ends (RACE)............................20 (9) Histochemical staining............................................21 (10) GenBank accession numbers........................................21 (11) High-performance liquid chromatography (HPLC) analysis...............22 Results.............................................................23 (1) Regeneration and transformation.....................................23 (2) Identification of transgenic plants....................................24 (3) Isolation and comparison of E. pallida CSH genes.......................25 (4) EpaCHS expression and metabolites analysis in CHS transgenic plants.......26 Discussion..........................................................27 (1) E. pallida regeneration............................................27 (2) E. pallida transformation...........................................28 (3) Characterization of E. pallida CHS genes..............................30 (4) Molecular characterization of transformants............................31 Tables.............................................................34 Figures.............................................................36 2. Chapter 2: Modification of flower color by suppressing β-ring carotene hydroxylase genes in Oncidium orchids...............................49 Abstract............................................................49 Introduction.........................................................50 Materials and Methods................................................54 (1) Plant materials...................................................54 (2) Cloning of BCH2 and plasmid construction............................54 (3) Transgenic identification and histochemical staining.....................55 (4) Rapid amplification of cDNA ends (RACE) and BD GenomeWalker Universal kit.............................................................55 (5) Phylogenetic analysis.............................................56 (6) Plant transformation..............................................56 (7) PCR DIG probe..................................................57 (8) High-performance liquid chromatography analysis......................58 Results.............................................................59 (1) Isolation and comparison of BCH genes...............................59 (2) Expression of BCH2 in Oncidium....................................59 (3) Identification of transgenic plants....................................61 (4) BCH2 expression in RNAi transgenic plants...........................62 (5) Pigment analysis of flowers........................................62 Discussion..........................................................64 (1) Characterization of Oncidium BCH genes.............................64 (2) Oncidium Transformation and molecular characterization of transformants...67 (3) HPLC method development, evaluation and validation...................68 Figures.............................................................70 3. Conclusion.......................................................82 Figures.............................................................85 References..........................................................88 Supporting information...............................................106 Tables............................................................106 Figures............................................................111 | |
dc.language.iso | en | |
dc.title | 淡花紫錐菊和文心蘭花色基因功能分析 | zh_TW |
dc.title | Functional studies of pigmentation genes in Echinacea pallida and Oncidium orchids | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 靳宗洛,林讚標,陶建英,張正,陳福旗 | |
dc.subject.keyword | 農桿菌轉殖,苯基苯乙烯酮合成?,β-胡蘿蔔素羥化?,高效液相層析, | zh_TW |
dc.subject.keyword | Agrobacterium-mediated transformation,chalcone synthase,β-ring carotene hydroxylase,High-performance liquid chromatography, | en |
dc.relation.page | 112 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-01-29 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 植物科學研究所 | zh_TW |
顯示於系所單位: | 植物科學研究所 |
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