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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 葉開溫 | |
dc.contributor.author | Peng-Jen Chen | en |
dc.contributor.author | 陳鵬仁 | zh_TW |
dc.date.accessioned | 2021-06-16T08:23:17Z | - |
dc.date.available | 2019-01-27 | |
dc.date.copyright | 2014-01-27 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-01-24 | |
dc.identifier.citation | Reference
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58636 | - |
dc.description.abstract | 葉綠體轉殖技術擁有多項優點,包含使轉基因具有高表達量及容易做多基因的構築,因此葉綠體轉殖技術被認為是次世代的轉基因技術。為了使轉殖株具有多重抵抗病原菌及蟲害的能力,我們從高麗菜的genomic DNA中釣取trnI/trnA flanking sequence作為葉綠體轉殖載體的骨幹並構築包含甘藷的sporamin基因, 芋頭的CeCPI 基因和來自Paecilomyces javanicus.的chitinase基因以上三個防禦基因的載體。利用基因槍轉殖法和抗生素持續篩選的方式,成功地得到若干株葉綠體菸草(N. benthamiana)轉殖株,這些轉植株都具有高表達的RNA及蛋白質表現層次。透過免疫金標記技術證實了轉基因大量累積表現在葉綠體轉殖株的葉及根之質體(plastid)中。將轉殖株的葉片餵食斜紋夜盜(Spodoptera litura)及甜菜葉蛾(Spodoptera exigua)兩種不同的昆蟲,發現轉殖株具有高度的抗蟲能力;同時實驗結果也證明轉殖株具有抵抗葉斑病真菌(Alternaria alternata)及軟腐病細菌(Pectobacterium carotovorum subsp. carotovorum)的能力。更進一步的研究發現轉殖株也具有抗旱及抗鹽的的能力。轉殖株可能透過CeCPI經由減少細胞膜氧化及細胞內離子流失之傷害,以達到抵抗非生物性逆境的能力。我們的實驗結果證實了同時轉殖三種不同的防禦基因在植物葉綠體中,各別基因可以透過協同作用的方式使植物具有抵抗多重生物性及非生物性逆境的能力。 | zh_TW |
dc.description.abstract | Plastid engineering provides several advantages for the next generation of transgenic technology, including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. With the goal of generating transplastomic plants with multi-resistance against both phytopathogens and insects, a construct containing a monocistronic patterned gene stack was transformed into N. benthamiana plastids harboring sweet potato sporamin, taro cystatin, and chitinase from Paecilomyces javanicus. Transplastomic lines were screened and characterized by Southern/Northern/Western blot analysis for the confirmation of transgene integration and respective expression level. Immunogold localization analyses confirmed the high level of accumulation proteins that were specifically expressed in leaf and root plastids. Subsequent functional bioassays confirmed that the gene stacks conferred a high level of resistance against both insects and phytopathogens. Specifically, larva of Spodoptera litura and Spodoptera exigua either died or exhibited growth retardation after ingesting transplastomic plant leaves. In addition, the inhibitory effects on both leaf spot diseases caused by Alternaria alternata and soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum were markedly observed. Moreover, tolerance to abiotic stresses such as salt/osmotic stress was highly enhanced. The CeCPI gene may be involved in reducing the cell membrane oxidation and ion leakage from cell to protect the plant from abiotic stress. The results confirmed that the simultaneous expression of sporamin, cystatin and chitinase conferred a broad spectrum of resistance. Conversely, the expression of single transgenes was not capable of conferring such resistance. To the best of our knowledge, this is the first study to demonstrate an efficacious stacked combination of plastid expressed defense genes which resulted in an engineered tolerance to various abiotic and biotic stresses. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T08:23:17Z (GMT). No. of bitstreams: 1 ntu-103-D96b42005-1.pdf: 12706999 bytes, checksum: 6531f5552e1fc1ef22f1008c2c346d03 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 中文摘要 2
Abstract 3 List of Figures 8 List of Tables 9 List of Abbreviations 10 1. Introduction 11 1.1 The concept of second generation transgenic plant 12 1.2 The advances and current studies in chloroplast engineering 12 1.3 Current studies of sporamin, CeCPI and chitinase 16 1.3.1 Sporamin 16 1.3.2 CeCPI (cysteine protease inhibitor) 18 1.3.3 Chitinase 19 1.4 Research objectives 20 2. Materials and methods 22 2.1 Plant material and growth condition 22 2.2 Construction of transformation vectors 22 2.3 Plastid transformation and selection of homoplasmic transplastomic N. benthamiana lines 23 2.4 Southern and Northern blot analyses 24 2.5 Protein activity assay 24 2.5.1 The proteinase inhibitor activity 24 2.5.2 Endo-chitinase activity assay 25 2.6 Immunolocalization of sporamin and CeCPI in transplastomic N. benthamiana plants 26 2.7 Western blot analysis for transplastomic N. benthamiana plants 27 2.8 Insect bioassays 28 2.9 Antibacterial assays 28 2.10 Antifungal assays 29 2.11 Salt and osmotic stress assay 29 2.12 Determination of oxidative damage to lipids 30 2.13 Electrolyte leakage assay 31 2.14 Statistical analysis 31 3. Results 32 3.1 Plastid expression vectors construction 32 3.2 Generation of transplastomic Nicotiana benthamiana plants 32 3.3 Molecular characterization of transplastomic plants 33 3.4 Protein expression, enzyme activity and immunolocalization 35 3.5 Insect resistance in transplastomic plants 36 3.6 Bacterial resistance in transplastomic plants 38 3.7 Fungal resistance in transplastomic plants 39 3.8 Abiotic stress tolerance in transplastomic plants 40 3.8.1 Salt and osmotic stress 40 3.8.2 Effect of methyl viologen (MV) and sodium chloride (NaCl) on leaf bleaching and oxidative damage to membrane lipids 41 4. Discussion 43 5. Future prospect 46 Figures 48 Tables 79 Reference 81 Appendix 91 Appendix 1. A protocol of plastid transformation in N. benthamiana 91 Appendix 1.1 Plant material preparation 91 Appendix 1.2 Coating gold particles with DNA 91 Appendix 1.3 Bombardment of leaf tissue 92 Appendix 1.4 Selection and regeneration of transplastomic plants 93 Appendix 2 The pine-tree method for RNA extraction 95 Appendix 3 The CTAB method for DNA extraction 97 Appendix 4 Publications 99 Appendix 4.1 Chen, P.J., Senthilkumar, R., Jane, W.N., He, Y., Tian, ZH and Yeh, K.W. (2014). Transplastomic Nicotiana benthamiana plants expressing multiple defense genes encoding protease inhibitors and chitinase display broad spectrum resistance against insects, pathogens, and abiotic stresses. Plant Biotechnol. J. (In press) 99 Appendix 4.2 Chang, I.F., Chen, P.J., Shen, C.H., Hsieh, T.J., Hsu, Y.W., Huang, B.L., Kuo, C.I., Chen, Y.T., Chu, H.A., Yeh, K.W., and Huang, L.C. (2010). Proteomic profiling of proteins associated with the rejuvenation of Sequoia sempervirens (D. Don) Endl. Proteome science 8, 64. 99 Appendix 4.3 Dong, S., Tian, Z., Chen, P.J., Senthil Kumar, R., Shen, C.H., Cai, D., Oelmüllar, R., and Yeh, K.W. (2013). The maturation zone is an important target of Piriformospora indica in Chinese cabbage roots. J. Exp. Bot. 64, 4529-4540. 100 Appendix 4.4 Ye, W., Shen, C.H., Lin, Y., Chen, P.J., Xu, X., Oelmüller, R., Yeh, K.W., and Lai, Z. (2014). Growth Promotion-Related miRNAs in Oncidium Orchid Roots Colonized by the Endophytic Fungus Piriformospora indica. PloS one 9, e84920. 100 | |
dc.language.iso | en | |
dc.title | 利用基因疊架法轉殖蛋白質抑制酶及幾丁質酶至菸草葉綠體中以產生多重抗性的轉基因作物 | zh_TW |
dc.title | Transplastomic Nicotiana benthamiana plants expressing multiple defense genes encoding protease inhibitors and chitinase display broad spectrum resistance against insects, pathogens, and abiotic stresses | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 陳文輝,楊文彬,曾顯雄,鄭秋萍,林學詩 | |
dc.subject.keyword | 葉綠體轉殖, | zh_TW |
dc.subject.keyword | sporamin,CeCPI,chitinase,gene stacking,plastid transformation,stress tolerance, | en |
dc.relation.page | 100 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-01-24 | |
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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