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軟腐菌篩選系統應用於蝴蝶蘭花粉塊基因轉殖植株篩選之研究 = Studie...

國立高雄大學生物科技研究所

軟腐菌篩選系統應用於蝴蝶蘭花粉塊基因轉殖植株篩選之研究 = Studies on Erwinia selection system with pollinia transformation to screen transgenic line of Phalaenopsis

紀錄類型:	書目-語言資料,印刷品 : 單行本
並列題名:	Studies on Erwinia selection system with pollinia transformation to screen transgenic line of Phalaenopsis
作者:	朱恩緯,
其他團體作者:	國立高雄大學
出版地:	[高雄市]
出版者:	撰者;
出版年:	2008[民97]
面頁冊數:	91面圖，表 : 30公分;
標題:	基因轉殖
標題:	Erwinia selection system
電子資源:	http://handle.ncl.edu.tw/11296/ndltd/05243967287377147480
附註:	指導教授：陳文輝、葛孟杰
附註:	參考書目：面66-73
附註:	附錄：pCAM1304-PeUFGT3載體DNA示意圖等7種
摘要註:	蝴蝶蘭為台灣重要的經濟花卉，是目前台灣四大外銷旗鑑農產品之一。在前人研究顯示在轉殖植株中大量表現 pflp ( sweet pepper ferredoxin-like protein ) 基因可增強作物對細菌性軟腐病原的抗性，You 等人( 2003 )曾利用 pflp 抗病基因來當做篩選標識基因 ( selection marker ) 選殖基因轉殖蘭花。但由先前篩選技術以 PLB以浸潤方式至軟腐菌篩選，誘發高度的過敏反應，進而導致植物計畫性細胞死亡( Programed cell death )現象。因此我們希望能改善軟腐菌篩選系統以葉片穿刺方法來篩選蝴蝶蘭轉殖株。本研究以台灣阿嬤蝴蝶蘭 ( Phalaenopsis Aphrodite subps formosana ) 當做試驗材料。在花粉塊基因轉殖結果顯示，以農桿菌處理之轉殖結莢率為56 %～70.8 %，以農桿菌配合基因槍處理之轉殖結莢率為60.3 %～79.1 %。軟腐菌篩選濃度為 5×104 cfu /ml，篩選後存活的轉殖植株分別為PeSOC1 (＋) 轉殖株可得到46.3 %軟腐菌篩選存活率，PeUFGT3 (＋) 轉殖株可得到 30.3%軟腐菌篩選存活率， PeSOC1(－) 轉殖株可得到 24.6 %軟腐菌篩選存活率。上述三種抗軟腐菌轉殖植株進行GUS染色分析，確認其基因是否表現，GUS表現所得的轉殖株成功率分別為PeSOC1(＋) 轉殖株可得到 29.8 %，PeUFGT3 (＋)轉殖株可得到30.2 %，PeSOC1(－) 轉殖株可得到15.5 %。初步將三種不同轉殖植株分別取有 GUS 表現轉殖植株抽取 genomic DNA進行 PCR 分析，結果顯示轉殖植株成功的轉殖入 pflp基因，使得植株具有抗病能力。綜合以上結果以軟腐菌葉片穿刺篩選方法相較前人篩選方法有較佳篩選效率。 Orchid is an economic flowersing crop. It is one of the most important agricultural products for export in Taiwan. Previous study indicated that PFLP (sweet pepper ferredoxin-like protein), an anti-microbial protein, might be able to control the plant diseases via reducing the growth of pathogen. A novel method for selection of transgenic plants utilizing the pflp gene as selection marker and Erwinia carotovora as the selection agent was developed. The Erwinia selection system does not olny solve traditional antibiotic problem but also acceletate the selection progress. In this study, we developed an Erwinia selection system with pollinia transformation to screen transgenic lines of Phalaenopsis. When the genes were first transformed into orchid pollinia via Agrobactium tumefaciens-mediated transformation, 56 %～70.8% capsule rates could be reached after pollination. When particle bombardment combined with A. tumefaciens-mediated transformation to transfer the genes into pollinia, 60.3 %～79.1 % capsule rates could be reached after pollination. The concentration of 5×104 CFU/ml infection suspension of E.carotovora was found to be a suitable threshold to screen transformed plants by injecting to the leaves. The survival rates of transformed plants after E. carotovora selection were 46.3 % in PeSOC (＋), 30.3 % in PeUFGT3 (＋), and 24.6 % in PeSOC1(－), individually. For identifying levels of gene expression GUS histochemical staining on transformed plants was performed. The GUS expression rates in transformed plants were 29.8 % in PeSOC(＋), 30.2 % in PeUFGT3 (＋), and 15.5 % in PeSOC1(－), individually. Through PCR analysis of the genomic DNA on some GUS-positive E. carotovora selection system resistant transgenic lines, the specific 0.6 kb DNA fragment of the pflp gene

軟腐菌篩選系統應用於蝴蝶蘭花粉塊基因轉殖植株篩選之研究 = Studies on Erwinia selection system with pollinia transformation to screen transgenic line of Phalaenopsis
朱, 恩緯

軟腐菌篩選系統應用於蝴蝶蘭花粉塊基因轉殖植株篩選之研究 = Studies on Erwinia selection system with pollinia transformation to screen transgenic line of Phalaenopsis / 朱恩緯撰 - [高雄市] : 撰者, 2008[民97]. - 91面 ; 圖，表 ; 30公分.
指導教授：陳文輝、葛孟杰參考書目：面66-73附錄：pCAM1304-PeUFGT3載體DNA示意圖等7種.
基因轉殖Erwinia selection system

軟腐菌篩選系統應用於蝴蝶蘭花粉塊基因轉殖植株篩選之研究 = Studies on Erwinia selection system with pollinia transformation to screen transgenic line of Phalaenopsis
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330 $a 蝴蝶蘭為台灣重要的經濟花卉，是目前台灣四大外銷旗鑑農產品之一。在前人研究顯示在轉殖植株中大量表現 pflp ( sweet pepper ferredoxin-like protein ) 基因可增強作物對細菌性軟腐病原的抗性，You 等人( 2003 )曾利用 pflp 抗病基因來當做篩選標識基因 ( selection marker ) 選殖基因轉殖蘭花。但由先前篩選技術以 PLB以浸潤方式至軟腐菌篩選，誘發高度的過敏反應，進而導致植物計畫性細胞死亡( Programed cell death )現象。因此我們希望能改善軟腐菌篩選系統以葉片穿刺方法來篩選蝴蝶蘭轉殖株。本研究以台灣阿嬤蝴蝶蘭 ( Phalaenopsis Aphrodite subps formosana ) 當做試驗材料。在花粉塊基因轉殖結果顯示，以農桿菌處理之轉殖結莢率為56 %～70.8 %，以農桿菌配合基因槍處理之轉殖結莢率為60.3 %～79.1 %。軟腐菌篩選濃度為 5×104 cfu /ml，篩選後存活的轉殖植株分別為PeSOC1 (＋) 轉殖株可得到46.3 %軟腐菌篩選存活率，PeUFGT3 (＋) 轉殖株可得到 30.3%軟腐菌篩選存活率， PeSOC1(－) 轉殖株可得到 24.6 %軟腐菌篩選存活率。上述三種抗軟腐菌轉殖植株進行GUS染色分析，確認其基因是否表現，GUS表現所得的轉殖株成功率分別為PeSOC1(＋) 轉殖株可得到 29.8 %，PeUFGT3 (＋)轉殖株可得到30.2 %，PeSOC1(－) 轉殖株可得到15.5 %。初步將三種不同轉殖植株分別取有 GUS 表現轉殖植株抽取 genomic DNA進行 PCR 分析，結果顯示轉殖植株成功的轉殖入 pflp基因，使得植株具有抗病能力。綜合以上結果以軟腐菌葉片穿刺篩選方法相較前人篩選方法有較佳篩選效率。 Orchid is an economic flowersing crop. It is one of the most important agricultural products for export in Taiwan. Previous study indicated that PFLP (sweet pepper ferredoxin-like protein), an anti-microbial protein, might be able to control the plant diseases via reducing the growth of pathogen. A novel method for selection of transgenic plants utilizing the pflp gene as selection marker and Erwinia carotovora as the selection agent was developed. The Erwinia selection system does not olny solve traditional antibiotic problem but also acceletate the selection progress. In this study, we developed an Erwinia selection system with pollinia transformation to screen transgenic lines of Phalaenopsis. When the genes were first transformed into orchid pollinia via Agrobactium tumefaciens-mediated transformation, 56 %～70.8% capsule rates could be reached after pollination. When particle bombardment combined with A. tumefaciens-mediated transformation to transfer the genes into pollinia, 60.3 %～79.1 % capsule rates could be reached after pollination. The concentration of 5×104 CFU/ml infection suspension of E.carotovora was found to be a suitable threshold to screen transformed plants by injecting to the leaves. The survival rates of transformed plants after E. carotovora selection were 46.3 % in PeSOC (＋), 30.3 % in PeUFGT3 (＋), and 24.6 % in PeSOC1(－), individually. For identifying levels of gene expression GUS histochemical staining on transformed plants was performed. The GUS expression rates in transformed plants were 29.8 % in PeSOC(＋), 30.2 % in PeUFGT3 (＋), and 15.5 % in PeSOC1(－), individually. Through PCR analysis of the genomic DNA on some GUS-positive E. carotovora selection system resistant transgenic lines, the specific 0.6 kb DNA fragment of the pflp gene
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