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標題: | 苦瓜植物生長素運送蛋白之過量表達與定位分析 Overexpression and localization of auxin transporters from Momordica charantia L. |
作者: | Yi-Wei Lin 林宜瑋 |
指導教授: | 杜宜殷(Yi-Yin Do) |
關鍵字: | 植物生長素運送蛋白,植物生長素極性運輸,植物發育,植物生長素差異性分布, auxin transporter,polar auxin transport,plant development,auxin differential distribution, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 苦瓜之果實發育受到植物生長素影響,而植物生長素主要藉由植物生長素運送蛋白在細胞膜上的不對稱分布而呈不同濃度分布,影響植物發育。分別使用過量表達苦瓜植物生長素輸出 (McPIN1、McPIN3) 及輸入 (McLAX1、McLAX2) 運送蛋白基因,及綠色螢光蛋白 (green fluorescence protein, GFP) 與苦瓜植物生長素運送蛋白融合基因 (McPIN1:GFP、McPIN3:GFP、McLAX1:GFP及McLAX2:GFP) 之重組質體,以農桿菌為媒介,轉殖入菸草及阿拉伯芥作為研究材料。菸草轉殖株經南方氏雜交分析確認嵌入基因拷貝數,並以聚合酶連鎖反應確認嵌入基因之完整性。過量表達McPIN1之菸草轉殖株葉片出現皺摺。過量表達綠色螢光蛋白與各苦瓜植物生長素運送蛋白融合基因之菸草轉殖株總花朵數較多,且過量表達McLAX1:GFP及McLAX2:GFP之轉殖株具較多分支。菸草轉殖株NtMcPIN3:GFP-10具強分支性,總花朵數也明顯多於未轉殖株及其他轉殖株。於阿拉伯芥葉片原生質體暫時性表達綠色螢光蛋白與苦瓜植物生長素運送蛋白融合基因,與細胞膜定位標準螢光蛋白mCherry表現位置一致,確認苦瓜植物生長素運送蛋白為膜蛋白。觀察過量表達McPIN3:GFP融合蛋白之菸草轉殖株中GFP分佈位置,得知苦瓜PIN3蛋白集中在根部軸柱細胞基部、莖部保衛細胞膜及周圍細胞基部以及盛開花朵的花藥表現。以GFP抗體進行菸草轉殖株NtMcPIN1:GFP-11及NtMcPIN3:GFP-10中融合蛋白免疫定位,結果顯示輸出運送蛋白累積於葉片表皮細胞、葉片維管束組織、發育中葉芽基部及表皮細胞。觀察表達McLAX1:GFP阿拉伯芥轉殖株,於根部表皮細胞及維管束鞘細胞、莖部維管組織及葉片尖端有目標蛋白表現。觀察過量表達McLAX2:GFP融合蛋白之菸草轉殖株中GFP分佈位置,得知苦瓜LAX2蛋白多於根尖表皮細胞及根部維管束鞘細胞內側與莖部皮層細胞內側表現。針對菸草轉殖株NtMcLAX1:GFP-12及NtMcLAX2:GFP-12進行融合蛋白免疫定位,結果顯示輸入運送蛋白累積於葉片表皮細胞、葉肉細胞、葉片維管束組織、頂芽及芽體表層細胞。 The fruit development of bitter gourd is affected by auxin. The differential distribution of auxin mainly via the asymmetric localization of auxin transporters on plasma membrane affects plant growth. Overexpression of bitter gourd auxin efflux (McPIN1 and McPIN3), and influx (McLAX1 and McLAX2) transporter genes, and GFP-fused (McPIN1:GFP, McPIN3:GFP, McLAX1:GFP and McLAX2:GFP) plasmids were transferred into tobacco and Arabidopsis through Agrobacterium-mediated transformation for analysis. The target gene copy number of tobacco transformants had been confirmed by Southern blot analysis and the gene integrity of target genes in transformants was confirmed by polymerase chain reaction. Tobacco transformants overexpressing McPIN1 exhibited wrinkled-leaf. The flower number of tobacco transformants for all bitter gourd auxin transporters and GFP fusion proteins is more than untransformed tobacco. Furthermore, the transformants overexpressing McLAX1:GFP and McLAX2:GFP produced more number of branches than untransformed control plant. NtMcPIN3:GFP-10 produced the most number of branches and flowers when compared to the untransformed plant and other transformants. By transient expression of bitter gourd auxin transporters and GFP fusion proteins in Arabidopsis mesophyll protoplast, the expression pattern of fusion proteins and plasma membrane mCherry marker were identical, and the bitter gourd auxin transporters are membrane proteins. By GFP fluorescence observation in tobacco transformants overexpressing McPIN3:GFP, McPIN3 proteins localized mainly on basal side of root stele cells, cell membrane of stem guard cells, basal side of cells along stem, and the anther of full bloom flowers. Immunolocalization of GFP in tobacco NtMcPIN1:GFP-11 and NtMcPIN3:GFP-10 transformants showed that fusion proteins accumulated on leaf epidermal cells, leaf vascular cells, basal side of leaf primordium and the adjacent cells. By GFP fluorescence observation in Arabidopsis transformants, McLAX1:GFP fusion proteins localize on root epidermal cells, bundle sheath cells, stem vascular cells and leaf tip cells. It indicated that McLAX2 proteins localized mainly on the epidermal cells in root tip, inner side of root bundle sheath cells, and inner side of stem cortex cells by GFP fluorescence observation in tobacco transformants overexpressing McLAX2:GFP. Immunolocalization of tobacco transformants NtMcLAX1:GFP-12 and NtMcLAX2:GFP-12 by GFP antibody showed that target proteins expressed on leaf epidermal cells, mesophyll cells, leaf vascular cells and the apical bud. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30251 |
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顯示於系所單位: | 園藝暨景觀學系 |
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