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  2. 農業暨自然資源學院
  3. 生物科技學研究所
Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90058
標題: Characterization and Functional Analysis of Genes in Regulating Flower Transition and Flower Senescence in Eustoma grandiflorum
洋桔梗中與開花時間及花朵老化相關基因之探討與功能性分析
作者: 李坤紘
Kun-Hung Li
關鍵字: 洋桔梗;開花時間;花器老化;簇生化;Eustoma grandiflorum;Flower Transition;Flower Senescence;rosette
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摘要: 
洋桔梗在國際市場上是相當重要的高經濟花卉。目前洋桔梗待解決的問題是於25°C以上高溫會導致簇生化而抑制開花，且二週的瓶插壽命限制了外銷的市場，因此本實驗目的是利用基因轉殖調控洋桔梗開花時間與瓶插壽命。前人研究中已知阿拉伯芥中的FT與LFY是促進開花基因，本實驗從洋桔梗中選殖出EgFT與EgLFY並進行功能性分析。實驗結果得知，EgFT主要表現在葉片與未成熟花苞中的花萼與花瓣。大量表現EgFT阿拉伯芥轉殖株藉由促進AP1基因表現而提早開花。大量表現EgFT洋桔梗轉殖株藉由促進MADS box基因中的A群基因EgAP1與EgFUL表現而在高溫生長環境中也能正常抽苔與開花。另外，大量表現EgFT洋桔梗轉殖株導致雄蕊與雌蕊轉變成花萼化花瓣，同時可偵測到MADS box基因中的A群基因：EgAP1、EgFUL與E群基因：EgSEP1、EgSEP3受到正調控，而C群基因EgAG受到負調控。以上結果顯示EgFT促進A與E群基因，並抑制C群基因而調控開花時間與花萼與花瓣之形成。EgLFY主要表現在未成熟花苞中的花萼。大量表現EgLFY阿拉伯芥轉殖株可觀察到頂芽分生組織轉變成花器之性狀，然而大量表現EgLFY洋桔梗轉殖株觀察到花器轉變形成花序分生組織或形成多花萼之結構。大量表現EgLFY洋桔梗轉殖株的A群基因EgAP1受到正向調控，而B群基因EgAP3、EgPI與 C群基因EgAG以及E群基因EgSEP3受到負調控。以上結果顯示EgLFY促進A群基因，並抑制B/C/E群基因而調控花萼之形成。前人研究已知阿拉伯芥中LFY與TFL1共同調控花序之結構，本實驗也選殖出洋桔梗中TFL1同源基因進行功能性分析。EgTFL1主要表現在未成熟花苞的花瓣、雄蕊及雌蕊，此表現量與EgLFY呈現負相關，同時也觀察到EgTFL1在大量表現EgLFY洋桔梗轉殖株中受到負調控，因此推測EgLFY與EgTFL1表現量可互相拮抗並分別調控洋桔梗花序與花器分生組織之形成。(第二章)
前人研究指出大量表現SVP與AGL24基因可延緩阿拉伯芥花器老化與凋落。為了增加洋桔梗瓶插壽命，本實驗從洋桔梗中選殖出SVP與AGL24同源基因並進行功能性分析。EgSVP主要表現在葉片與未成熟花苞中的花萼、花瓣以及雌蕊; EgAGL24主要表現在成熟花器中的花瓣以及在葉片也可偵測到表現量。大量表現EgSVP與EgAGL24在阿拉伯芥中可觀察到晚開花性狀以及下游的FT受到負調控。在阿拉伯芥中大量表現EgSVP與EgAGL24有延緩花器老化與凋落之性狀，且此花器不老化與不凋落之性狀並不受到乙烯處理的影響。利用即時相對定量分析測定基因表現量，乙烯路徑基因ERF1與離層脫落起始相關基因IDA、HAESA以及HSL2受到抑制。綜合以上結果得知，EgSVP與EgAGL24藉由調控乙烯訊息路徑與離層脫落起始相關基因，進而調節阿拉伯芥花器的老化與凋落。(第三章)

Eustoma grandiflorum is an important economic flower crop in the international market. Two srious problems for E. grandiflorum floral industry is the rosetting under higher temperature (> 25°C) and the short vase life for only two weeks. The aim in this study is to manipulate flowering time and vase life of E. grandiflorum by gene transformation. It has been reported that FT and LFY are activators for flowering. EgFT and EgLFY were isolated and functionally analyzed in this study. EgFT was predominantly expressed in leaf and the sepal and petal of young flower buds. Early flowering was observed in the 35S::EgFT transgenic Arabidopsis by upregulating the expression of AP1. 35S::EgFT transgenic Eustoma bypassed rosetting and bolted under high-temperature conditions by upregulating of A (EgAP1/EgFUL) functional genes. In addition, stamen and carpel were converted into sepal/petal like structures due to the up-regulation of A (EgAP1/EgFUL), E (EgSEP3/EgSEP1) and down-regulation of C (EgAG) functional genes in 35S::EgFT Eustoma. These results indicated that EgFT is able to promote flowering and regulate sepal/petal formation by activating the A/E genes and suppressing the C gene in E. grandiflorum. EgLFY was predominantly expressed in the sepal of young flower buds. 35S::EgLFY converted shoot meristems into flowers in the transgenic Arabidopsis. Interestingly, floral meristems were converted into inflorescence meristems or developed into numerous sepals in 35S::EgLFY transgenic Eustoma. The upregulation of A (EgAP1) and downregulation of B (EgAP3/EgPI), C (EgAG) and E (EgSEP3) functional MADS box genes were observed in 35S::EgLFY Eustoma. These results indicated that EgLFY is able to regulate sepal formation by activating the A genes and suppressing the B/C/E gene in E. grandiflorum. In this study, EgTFL1 was also isolated and functionally analyzed. EgTFL1 was predominantly expressed in the petal, stamen and carpel of young flower buds, and the expression pattern was opposite to EgLFY. In addition, the expression level of EgTFL1 was downregulated in the 35S::EgLFY Eustoma. These results indicated that the EgLFY and EgTFL1 antagonized each other to regulate the development of inflorescence and floral meristems. (Chapter 2)
It has been reported that ectopic expression of Arabidopsis MADS box genes SVP and AGL24 delay flower senescence and abscission in transgenic Arabidopsis. In order to improve vase life for E. grandiflorum flowers, two homologous genes of Arabidopsis SVP and AGL24 were isolated and characterized from E. grandiflorum. EgSVP was highly expressed in leaf and the sepal/petal/carpel of young flower buds. EgAGL24 mRNA was also detected in leaf and was predominantly expressed in petal of the mature flowers. Flowering time was delayed and the expression of downstream gene FT was repressed in 35S::EgSVP and 35S::EgAGL24 transgenic Arabidopsis. Furthermore, ectopic expression of EgSVP and EgAGL24 significantly delayed flower senescence and abscission in transgenic Arabidopsis. The delayed senescence and abscission of the flower organs in 35S::EgSVP and 35S::EgAGL24 Arabidopsis were unaffected by the ethylene treatment. Ethylene signaling pathway gene ERF1 and abscission initiation genes IDA, HAESA, HSL2 were repressed in 35S::EgSVP and 35S::EgAGL24 transgenic Arabidopsis. These data suggested a novel role for EgSVP and EgAGL24 in controlling the floral senescence and abscission by regulating ethylene perception and abscission initiation in Arabidopsis. (Chapter 3)
URI: http://hdl.handle.net/11455/90058
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