https://scholars.lib.ntu.edu.tw/handle/123456789/91570
標題: | 苦瓜植物生長素運送蛋白之功能分析與定位 Functional analysis and localization of auxin transporters in Momordica charantia L. |
作者: | 施宛昀 Shih, Wan-Yun |
關鍵字: | 植物生長素運送蛋白;極性運輸;細胞膜;綠色螢光蛋白;南方氏雜交分析;基因槍;掃描式共軛焦顯微鏡;auxin transporters;polar transport;cell membrane;green fluorescent protein;Southern analysis;gene gun;scanning confocal microscopy | 公開日期: | 2009 | 摘要: | 苦瓜為台灣重要的果菜類蔬菜,其不同階段的生長發育受到植物生長素的調控,而植物生長素於植物細胞間之運輸與分布受植物生長素運送蛋白影響。本研究分別構築過量表現正義與反義基因之苦瓜植物生長素輸入或輸出運送蛋白 (McLAX1、McLAX2、McPIN1與McPIN3) 轉殖質體,並以農桿菌媒介法轉殖入菸草,經由GUS 活性組織化學染色法及南方氏雜交分析,確認得到CaMV35S::McPIN3 與 CaMV35S::anti-McPIN3 菸草轉殖株各五株,並以反轉錄聚合酶連鎖反應與北方雜交分析,確認轉殖株皆有McPIN3基因的表現。另外,調查植株的生長勢,無論株高、葉片乾鮮重或根長,轉殖株都較佳,且都較早開花。以流式細胞儀分析葉片細胞週期的變化,得知 CaMV35S::McPIN3 與 CaMV35S::anti-McPIN3 菸草轉殖株的細胞分裂都比未轉殖株旺盛。利用徒手切片切取未轉殖株與轉殖株 (CaMV35S::McPIN3 與 CaMV 35S::anti-McPIN3) 菸草的莖部橫切面,於解剖顯微鏡下可觀察到轉殖株的維管束組織細胞數目較未轉殖株多。將融合綠色螢光蛋白之苦瓜植物生長素運送蛋白表現質體以基因槍槍擊至洋蔥表皮細胞後,可於掃描式共軛焦顯微鏡下觀察到苦瓜植物生長素運送蛋白於細胞膜上表現。而含有融合綠色螢光蛋白之苦瓜植物生長素運送蛋白菸草轉殖株之根部,於掃描式共軛焦顯微鏡下,則可觀察到 McPIN1 於根部的側根冠、軸柱、表皮、皮質、內皮、周皮層、維管束與靜止中心之細胞膜表現;McPIN3 蛋白則於根部之表皮、皮質、內皮細胞、周皮層與維管束組織表現,且綠色螢光蛋白於細胞的近基部端表現較明顯;苦瓜植物生長素輸入運送蛋白 McLAX1 於內皮、皮層、維管束組織與靜止中心的細胞膜上表現,而苦瓜植物生長素輸入運送蛋白 McLAX2 則於表皮、內皮、皮層與維管束細胞膜上表現。 Bitter gourd is one of the most important vegetables in Taiwan whose growth and development are mediated by plant hormone auxin. Recent studies showed that the distributions and transportation of auxin between cells are affected by the auxin carriers. Plasmids for over-expression and antisense auxin carriers from bitter gourd were constructed. Subsequently, the plasmids were introduced into tobaccos by Agrobacterium-mediated transformation. Each five transgenic plants for 35S::McPIN3 and 35S::anti-McPIN3 were confirmed by GUS staining and Southern analysis. McPIN3 was expressed in transgenic tobaccos analyzed by reverse transcription polymerase chain reaction and Northern hybridization. In contrast to the untranformed plant, the height, weight of fresh leaves and root length in transgenic plants were all much heiger. Besides, the transgenic plants bloomed earlier. The cell divisions of transgenic tobacco plants were much more active than the untranform tobacco plants by flow cytometry analysis. Forthermore, the cell numbers of vascular bundles in shoot of transgenic tobacco through transverse section were more than the untranformed tobacco observed under the anatomic microscopy. After gene gun transformation with gene fusion plasmid, green fluorescence was observed on the plasma membrane in onion epidermal cells under the scanning confocal microscopy. The cell membrane in lateral root cap, cortex, epidermis, endodermis, vasculature, columella and quenscent centre ofransgenic tobaccos carring the auxin efflux transporters McPIN1 expressed green fluorescence proteins examined under scanning confocal microscopy. Auxin efflux carriers McPIN3 expressed on the cell membranes of epidermis, endodermis, columella, cortex and vasculature of transgenic tobaccos. Auxin influx carriers McLAX1 localized on the cell membranes of endodermis, cortex, vasculature and quenscent centre. Auxin influx carriers McLAX2 expressed on the cell membranes of endodermis, cortex, vasculature and epidermis. Moreover, the strengths of green fluorescence in transgenic plant root are much stronger in apical and basal cell membrane than the lateral layers which may due to the polar localization of auxin transporters. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/181022 |
顯示於: | 園藝暨景觀學系 |
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ntu-98-R96628106-1.pdf | 23.53 kB | Adobe PDF | 檢視/開啟 |
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