苦瓜（Momordica charantia L.）植物生長素輸出運送蛋白基因之啟動子活性分析

Abstract

為瞭解苦瓜果植物生長素輸出運送蛋白(auxin efflux carrier)基因之啟動子活性及與其果實長發育的關係，將苦瓜植物生長素輸出運送蛋白基因McPIN1與McPIN3之啟動子表達載體，利用農桿菌媒介法轉殖至菸草，所得轉殖株進行GUS活性組織化學染色與誘導性試驗。結果顯示，McPIN1::GUS啟動子轉殖株在葉基部、側根尖可偵測到GUS表現，進一步觀察幼苗時期根部的啟動子活性，結果顯示McPIN1啟動子在根尖各構造皆有活性，其中以根冠與根分生組織活性最強，側根突出於內皮層時也可明顯見其活性表現；McPIN3啟動子於幼葉葉基部、葉肉、生長旺盛的根部與根尖、雌蕊具有活性表現，經切片利用顯微鏡觀察根部的GUS活性組織染色反應，McPIN3啟動子在皮層與中柱具有活性表現，並以中柱活性最強。誘導因子試驗方面，結果顯示NAA 、IBA、2,4-D、乙烯、100 mM ABA、低溫、高溫、光、重力與乾旱等處理，皆可促進McPIN1與McPIN3啟動子在不同組織的表現活性。相對的BA、kinetin、zeatin、20 mM ABA、GA3、水楊酸、MeJA與創傷處理則是抑制McPIN1和McPIN3啟動子使其活性表現變弱；McPIN3啟動子在葉部的活性表現對於誘導物的反應敏感性強，不若McPIN1啟動子在葉基部與根尖的活性誘導反應，可能受植株當時生長狀態影響較為曖昧難判斷是否因誘導物產生活性的改變，雖然兩啟動子對於保守性序列上的誘導因子的試驗各組織之感受能力不同，但誘導物對於兩啟動子活性是為正向促進或負向抑制的效果大致相符，顯示此兩基因家族蛋白對外界環境變化有共同合作應對的可能性。

To understand the promoter activities of auxin efflux carrier gene from bitter ground, promoter fragments of two auxin efflux carrier genes, McPIN1 and McPIN3, were transformed into tobacco for analysis. GUS staining was observed in the vein, the basal of leaf , roots and lateral roots of McPIN1::GUS transgenic tobaccos especially in dark color in root cap and root meristem. GUS expression was detected in the leaf, roots, the basal of leaf, and pistil of McPIN3::GUS transgenic tobaccos. The most part of McPIN3 promoter activity in root tip was performed in cortex. The promoter activities could be increased by NAA , IBA, 2,4-D, ethylene, 100 mM ABA, stress of temperate, tropism and drought treatments, and be decreased by BA, kinetin, zeatin, 20 mM ABA, GA3, SA, MeJA, and wounding. The response of McPIN3 promoter activity in leaf to inducements is more sensitive than McPIN1 promoter activity in the basal of the leaf and root tip, which might be affected by growth condition. Whereas there was differential sensation between McPIN1 and McPIN3 promoter activity, they had same trend up or down of the promoter acitivity.