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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王俊能(Chun-Neng Wang) | |
dc.contributor.author | Wen-Hsi Kuo | en |
dc.contributor.author | 郭聞喜 | zh_TW |
dc.date.accessioned | 2021-05-15T17:51:22Z | - |
dc.date.available | 2016-03-13 | |
dc.date.available | 2021-05-15T17:51:22Z | - |
dc.date.copyright | 2015-03-13 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2015-01-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5055 | - |
dc.description.abstract | 大岩桐(Sinningia speciosa)是著名的園藝植物,野生大岩桐具兩側對稱的花型;然而園藝栽培種中,輻射對稱花型的品種因大而美麗的花型而被保留並且大量地栽培。在模式植物金魚草中,CYCLOIDEA 是調控花朵兩側對稱發育的關鍵基因。在實驗室先前的研究中,發現 CYCLOIDEA 的同源基因(SsCYC)在輻射對稱花型品系中有一個小片段的核酸缺失(ΔSsCYC),很可能是造成兩側對稱花型發育模組喪失而轉變成為輻射對稱花型的原因。本研究將兩側對稱花型大岩桐的SsCYC基因轉殖進入菸草進行過量表現,和已知CYC基因能影響細胞面積、數目、調控器官大小的功能類似:菸草的花朵長度縮短、開口直徑下降、增加側芽、葉片蜷曲和植株的矮化。而過量表現有輻射對稱花的 ΔSsCYC,沒有性狀改變,代表核酸缺失使基因功能喪失。本研究也探討了大岩桐的組織誘導再生技術和農桿菌的感染條件,以期建立一個高效且穩定的基因轉殖系統。實驗結果顯示,當 MS 培養 基 中 擁 有 0.1 ppm naphthalene-acetic acid (NAA) 和 1 ppm 的6-benzylaminopurine (BA)時,會有最佳的再生率:以 6 mm 直徑葉切片為材料時可以獲得 86%的再生率,以 5 mm 長度的葉柄切塊為材料時可以獲得 56%的再生效率。更進一步地,本研究發現葉柄切塊在培養基上的擺放方向對於再生率有很大的影響,只有倒立或是水平放置的葉柄切塊可以成功地再生,推測可能和原本葉柄內部生長素向基性的分布模式有關。另外,本研究也發現再生的芽起源於葉片深處單一的維管束薄壁細胞,這和其他苦苣苔物種的起源於表皮細胞或是球型毛絨基座細胞不同。為了要找到最佳的農桿菌感染條件,本研究利用 GUS 報導基因的表現作為成功感染的標記,結果發現年輕的幼苗相較於成熟的葉切片或是葉柄切塊有較佳的感染效果,尤其是其子葉和第一對初生葉片,顯示大岩桐的幼苗相當有潛力發展成為良好的農桿菌基因轉殖材料。進一步透過石蠟切片發現感染的位置為表皮、葉肉和球型絨毛的頭狀細胞,然而這些組織是否可以誘導成再生苗仍有待進一步的實驗。另外,大岩桐的癒傷組織可能也是良好的基因轉殖材料。本研究為大岩桐 SsCYC 基因如何影響花部對稱的功能做了初步的探討。 | zh_TW |
dc.description.abstract | The native varieties of Sinningia speciosa (Gesneriaceae) bear zygomorphic flowers, but in horticultural varieties, large size showy actinomorphic flowers are selected due to human’s preference. CYCLOIDEA has been demonstrated to have a major genetic control in zygomorphy in Antirrhinum. In actinomorphic varieties, we found a small fragment deletion in its CYCLOIDEA homologue (∆SsCYC), which might indicate that the reversal to actinomorphy is a SsCYC loss of function mutant. I introduced CYC homologues from both zygomorphic cultivar (SsCYC) and actinomorphic cultivar (ΔSsCYC) into Nicotiana benthamiana, a closely related species to Sinningia speciosa, to verify whether ΔSsCYC has any effect on floral phenotype. I found that ectopic expression of SsCYC causes shorter longitudinal length of flowers, smaller floral opening diameters, induction of axillary shoots, curled leaves and dwarfism, agreed with CYC’s putative effects on cell proliferation or expansion. However, no visible phenotypic change could be observed in ΔSsCYC overexpression lines. I also optimized the genetic transformation system in Sinningia speciosa, focusing on tissue regeneration and Agrobacterium infection conditions. The MS medium supplied with 0.1 ppm naphthalene-acetic acid (NAA) and 1 ppm 6-benzylaminopurine (BA) was the best for shoot regeneration in both leaf and petiole explants. Eighty six percent and 56% regeneration rates were obtained from 6 mm diameter leaf explants and 5 mm petiole explants, respectively. Moreover, the orientation of petiole explants must be up-side down or horizontal to induce the shoot regeneration, which might relate to the endogenous basipetal distribution of auxin inside the petiole vascular tissue. It was found that the regenerative shoots of explants initiated from a single vascular parenchyma cell deep inside the regenerated tissue. This is different from other reported cases in Gesneriaceae species, in which their regenerative shoots usually originated from an epidermis cell or a glandular trichome basal cell. To explore whether Agrobacterium infection can enter regenerative tissue, transient transformation using GUS reporters was applied. In contrast to mature leaf or petiole explants, I found that young seedlings, especially those in cotyledonary stage or with the first pair of primary leaves have much higher success of transformation. This opens an opportunity that young seedlings are potential material for transformation. By paraffin sections, the positive signals of transformation were seen in epidermis, mesophyll and glandular trichome head cells but not inside the regenerative shoots. Alternatively, the induced callus tissue might be a better transformation material. This study provides a preliminary study on the functions of SsCYC genes and guidelines for further optimization of transformation system of Sinningia speciosa. | en |
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dc.description.tableofcontents | 致謝................................................................................................................................ I
中文摘要...................................................................................................................... III Abstract ........................................................................................................................ IV Content ......................................................................................................................... VI Index of Tables and Figures .......................................................................................... X Abbreviations ............................................................................................................ XIII Introduction .................................................................................................................... 1 Floral symmetry and its role in angiosperms diversification ................................. 1 Genetic mechanism of floral symmetry ................................................................. 1 Evolutionary history and conserved dorsal expression patterns of CYC-like genes among eudicots and monocots ............................................................................... 2 Functional divergence between CYC’s homologues ..................................... 4 Derived actinomorphy from zygomorphic ancestors ..................................... 6 Sinningia speciosa as a good model to study the genetics of floral symmetry...... 8 Functional analysis of SsCYC and ΔSsCYC in tobacco ............................. 12 Tissue culture and genetic transformation system of Sinningia speciosa .... 12 Aim of this study .................................................................................................. 15 Materials and Methods ................................................................................................. 16 Plant material and growth conditions .................................................................. 16 Sequence and phylogenetic analyses of the SsCYC gene .................................... 16 Southern blotting .................................................................................................. 17 Isolation of the upstream sequences of the SsCYC and ∆SsCYC genes ............... 18 VII Promoter analysis of the SsCYC and ∆SsCYC genes ........................................... 22 Vector construction for the functional analysis of the SsCYC and ∆SsCYC genes .............................................................................................................................. 22 Transformation of Nicotiana benthamiana leaf disk ........................................... 24 Preparation of aseptic tobacco seedling ....................................................... 24 Leaf disk infection ....................................................................................... 25 Selection of transformed shoots ................................................................... 26 Clearing method for measuring the cell size........................................................ 27 Genomic DNA extraction .................................................................................... 27 Total RNA extraction and reverse transcription ................................................... 29 Polymerase chain reaction ................................................................................... 30 Preparation of aseptic seedlings for tissue culture ............................................... 31 Tissue culture of Sinningia speciosa .................................................................... 32 Histological analysis by paraffin sections ............................................................ 33 Scanning electron microscope (SEM) ................................................................. 33 Transient transformation using GUS reporter gene ............................................. 34 Statistical analysis ................................................................................................ 37 Results .......................................................................................................................... 38 Conserved domains of the SsCYC gene ............................................................... 38 Reconstruction the phylogenetic tree of SsCYC gene .......................................... 45 Confirming the single copy of SsCYC gene in Sinningia speciosa ..................... 47 Isolation the upstream and coding sequences of SsCYC and ΔSsCYC ................ 49 Comparison of upstream sequence between SsCYC and ΔSsCYC ............. 49 Comparison of coding sequence between SsCYC and ΔSsCYC ................. 50 Construction the overexpressed SsCYC and ΔSsCYC transgenic lines ................ 54 Abnormal phenotypes among SsCYC transgenic plants ...................................... 61 Floral morphology measurement ................................................................. 66 Association between phenotypic abnormalities and mRNA expression levels ...................................................................................................................... 70 Floral tube cell shape and size measurements ............................................. 74 Tissue culture conditions for shoot induction in Sinningia speciosa ................... 78 Effects of petiole explant orientation on shoot regeneration ............................... 83 Callus induction from embryo ............................................................................. 86 Histological observation of shoot regeneration ................................................... 88 Scanning electron microscope observation .......................................................... 92 Testing the optimal explants source for Agrobacterium-mediated transformation .............................................................................................................................. 94 Discussion .................................................................................................................... 98 SsCYC is a homologue to AmCYC by sharing domains and phylogeny .............. 98 ΔSsCYC appears to lose its function due to a small fragment deletion ............... 99 ΔSsCYC gene is selected by human for its derived actinomorphy .................... 100 SsCYC represses the primary growth but promote the development of axillary buds ............................................................................................... 101 The downstream pathway of SsCYC is different from AmCYC ............... 103 SsCYC might cause male and female sterility in the plants with severe phenotypes ................................................................................................. 105 The results of functional analyses coincide with the genetic approach ............. 106 Shoot regeneration has two hormone dependent pathways ............................... 107 Orientation and size of petiole explants have effects on the regeneration rate .. 109 Shoots regeneration of Sinningia speciosa through direct embryogenesis ........ 110 Meristem identities are unstable in the newly regenerative shoots.................... 112 Young seedlings or callus might be a good source for transformation .............. 112 Conclusion and Future Prospects ............................................................................... 114 References .................................................................................................................. 116 Index of Tables and Figures Table 1 Genes used in construction of phylogenetic tree (Figure 2) ........................... 41 Table 2 Predicted trans-acting elements with known functions at the upstream regions of SsCYC and ∆SsCYC. ................................................................................................ 52 Figure 1 Floral morphologies of Sinningia speciosa .................................................. 10 Figure 2 Conserved domains and phylogeny of the SsCYC gene ............................... 44 Figure 3 Phylogeny of the SsCYC gene ...................................................................... 46 Figure 4 Southern blotting of detecting the copy number of SsCYC gene in Avanti (horticulture) and Espirito Santo (wild type) cultivars ................................................ 48 Figure 5 Illustration of SsCYC and ΔSsCYC genes and their putative promoters ....... 53 Figure 6 T-DNA insertion in T 0 transgenic populations .............................................. 57 Figure 7 mRNA expression levels of SsCYC or ΔSsCYC in T 0 transgenic lines ........ 59 Figure 8 Mature T 0 transgenic plant morphology ....................................................... 60 Figure 9 Different degrees of abnormality among the SsCYC transgenic plants ........ 62 Figure 10 Promoting the growth of axillary buds in SsCYC transgenic plants ........... 63 Figure 11 Leaf morphology of T 0 transgenic plants .................................................... 65 Figure 12 Flower morphology of T 0 transgenic plants at fully elongated stage ......... 67 Figure 13 Flower morphology measurement of SsCYC, ΔSsCYC transgenic tobacco plants. ........................................................................................................................... 69 Figure 14 Relative SsCYC mRNA expression level among T 0 population ................. 71 Figure 15 Flower morphology according to the relative mRNA expression level in SsCYC transgenic plants .............................................................................................. 73 Figure 16 Cell shape and size of floral tube measurement of SsCYC and ΔSsCYC transgenic tobacco plants ............................................................................................. 77 Figure 17 Effect of cytokinin and auxin concentration and explant sources on regeneration rate and shoots per responsive explant.................................................... 80 Figure 18 Effect of cytokinin and auxin concentration and explant sources on the morphology of shoot regeneration ............................................................................... 82 Figure 19 Effects of petiole explant orientations on shoot regeneration rate and shoots per responsive explant.................................................................................................. 84 Figure 20 Effects of petiole explant orientations on the morphology of regeneration ... ...................................................................................................................................... 85 Figure 21 Callus inductions from young seedlings ..................................................... 87 Figure 22 Process of shoot regeneration from leaf explants by paraffin section and dissecting microscope .................................................................................................. 91 Figure 23 Micro-scale observation of shoot regeneration by scanning electronic microscope ................................................................................................................... 93 Figure 24 Tissue competence for Agrobacterium infection by GUS transient assay .. 97 Supplementary Table 1 Arbitrary Degenerative Primer .......................................... 124 Supplementary Table 2 Putative transcription factor binding sites of SsCYC/ΔSsCYC .................................................................................................................................... 125 Supplementary Table 3 Primer list in this study ...................................................... 128 Supplementary Table 4 Flower morphology measurement of SsCYC and ΔSsCYC transgenic tobacco plants ........................................................................................... 130 Supplementary Table 5 Flower morphology according to the relative mRNA expression level in SsCYC transgenic plants ............................................................. 131 Supplementary Figure 1 Destination Vector pK2GW7,0 ........................................ 132 Supplementary Figure 2 Amino acid alignment of CYC-like gene for reconstruction the phylogeny ............................................................................................................. 133 Supplementary Figure 3 Promoter regions of SsCYC and ∆SsCYC genes ............. 135 Supplementary Figure 4 Nucleotide sequence of SsCYC and ∆SsCYC genes ........ 138 Supplementary Figure 5 Amino acid sequence of SsCYC and ∆SsCYC genes ....... 139 Supplementary Figure 6 Equal variance and normality tests of the floral length of three transgenic populations ...................................................................................... 140 Supplementary Figure 7 Equal variance and normality tests of the floral length of different mRNA expression levels among p35S::SsCYC::c-Myc transgenic population .................................................................................................................................... 141 Supplementary Figure 8 Preliminary result of Southern blotting of T 0 transgenic plants .......................................................................................................................... 142 | |
dc.language.iso | en | |
dc.title | 大岩桐SsCYC在圓葉菸草的表現及性狀觀察與其組織培養再生系統之優化 | zh_TW |
dc.title | Ectopic expression of SsCYC in Nicotiana benthamiana and optimizing regeneration system of Sinningia speciosa | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-1 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 洪傳揚(Chwan-Yang Hong) | |
dc.contributor.oralexamcommittee | 蔡新聲(Hsin-Sheng Tsay),陳仁治(Jen-Chih Chen) | |
dc.subject.keyword | 大岩桐,對稱性,CYCLOIDEA,組織培養,基因轉殖,農桿菌, | zh_TW |
dc.subject.keyword | Sinningia speciosa,symmetry,CYCLOIDEA,tissue culture,genetic transformation,Agrobacterium, | en |
dc.relation.page | 143 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2015-01-13 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生態學與演化生物學研究所 | zh_TW |
顯示於系所單位: | 生態學與演化生物學研究所 |
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