請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58636
標題: | 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 Transplastomic Nicotiana benthamiana plants expressing multiple defense genes encoding protease inhibitors and chitinase display broad spectrum resistance against insects, pathogens, and abiotic stresses |
作者: | Peng-Jen Chen 陳鵬仁 |
指導教授: | 葉開溫 |
關鍵字: | 葉綠體轉殖, sporamin,CeCPI,chitinase,gene stacking,plastid transformation,stress tolerance, |
出版年 : | 2014 |
學位: | 博士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58636 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-103-1.pdf 目前未授權公開取用 | 12.41 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。