苦瓜CTR1基因及其啟動子活性分析

Analysis of Constitutive Triple Response 1 Gene and its Promoter Activity in Bitter Gourd

陳慧玲(Hui-Ling Chen)

指導教授 ： 杜宜殷

共同指導教授 ： 黃鵬林(Pung-Ling Huang)

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10479

摘要

為分析苦瓜 (Momordica charantia L.)之CTR1 (Constitutive Triple Response 1)同源基因McCTR1之功能與特性，進行功能互補分析，將CaMV 35S::McCTR1轉殖質體，以農桿菌花序浸染法轉殖至阿拉伯芥突變株ctr1-1中，轉殖株外表型可恢復為野生型態大小。過量表達McCTR1基因之CaMV 35::McCTR1轉殖菸草，經GUS活性及南方氏雜交分析顯示，McCTR1基因已嵌入菸草基因組，並觀察到轉殖植株有瓶內開花的情形，CaMV 35S::McCTR1轉殖株之成熟植株較未轉殖菸草矮化並且會提早開花，但不易具有稔性。另外，CaMV 35S::McCTR1阿拉伯芥轉殖植株之外表形態較野生型小，根皆較野生型短；而黑暗中幼苗之胚軸及根較野生型長，但處理乙烯前趨物ACC後，轉殖株及未轉殖野生型均與突變株ctr1-1形態無顯著差異，顯示過量表達McCTR1基因之轉殖植株仍對乙烯具敏感性。為瞭解苦瓜McCTR1基因啟動子之活性，首先以南方氏雜交分析，確認McCTR1::GUS轉殖菸草，接著進行組織化學染色分析，結果顯示GUS活性表現在轉殖植株之莖段及根部組織。阿拉伯芥McCTR1::GUS轉殖植株AtP2發育初期GUS表現於根部及第一對本葉，而發育後期轉殖株之下位葉及根部皆有顯著的GUS呈色。以BA、 ABA、GA3及

關鍵字

乙烯 ； 苦瓜CTR1基因 ； 功能互補性 ； 過量表達 ； 啟動子活性分析

並列摘要

The CTR1 (constitutive triple response 1) ortholog (McCTR1) and its promoter activity from Momordica charantia L. have been characterized. Complementation of the Arabidopsis ctr1-1 mutant with the bitter gourd CTR1 gene regained like wild-type phenotype. CaMV 35S::McCTR1 transgenic tobaccos has been confirmed by GUS activity and Southern analysis indicating that McCTR1 located in tobacco genome. CaMV 35S::McCTR1 transgenic tobaccos accompanying with in vitro flowering, and in vivo were characterized by early flowering, hardly fertile, and shorter than wild-type of growth in the greenhouse. CaMV 35S::McCTR1 transgenic Arabidopsis plants have smaller size, shorter root in the light, but hypocotyls and roots longer than wild-type in the dark, while they showed no difference after 1-aminocyclopropane-1- carboxylic acid (ACC) treatment suggesting McCTR1-overexpressing plants were still sensitive to ethylene. For better understanding of McCTR1 promoter activity, Southern analysis and histochemical stain were used to verify McCTR1::GUS transgenic tobaccos which revealed GUS activity in stem and root. Besides, McCTR1::GUS transgenic Arabidopsis also had significant GUS activities in root and primary leaf during early developmental phase, and in old leaf and root during later developmental phase. The promoter activity of McCTR1::GUS Arabidopsis can be activated by BA, ABA, GA3, and SA treatment displaying GUS signal in old leaf. Various tissues can be observed for GUS activity by treatment of MeJA in root, NAA, 2,4-D in root tip, and ethylene in old leaf and root. Galactose, mannitol and trehalose can induce GUS activity in whole leaves. GUS stain faded in root using sorbitol or in the dark treatment. Heat induced McCTR1 expression in primary leaf and root, while induction of McCTR1 expression by wound was not significant.

並列關鍵字

ethylene ； McCTR1 ； ctr1-1 ； overexpression ； promoter activity

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