謝旭亮Hsieh, Hsu-Liang臺灣大學:植物科學研究所吳佩芸Wu, Pei-YunPei-YunWu2010-05-112018-07-062010-05-112018-07-062009U0001-0202200915223300http://ntur.lib.ntu.edu.tw//handle/246246/181941“光”是影響植物生長發育的重要環境因子之一。植物中可利用不同的光接受體(photoreceptor)如phytochromes(光敏素)，cryptochromes(隱花色素)和phototropins(植物向光素)來感應光環境的變化，包括光的方向性、週期、光量與光質。除此之外，Jasmonic acid (JA)已被認為一種植物荷爾蒙，參與在阿拉伯芥的生長發育與防禦機制之中，然而在植物體中如何整合光和JA的訊息傳遞仍有待研究。實驗室所研究的FIN219 (far-red insensitive 219)基因，參與在遠紅光上游的訊息傳遞，扮演著phyA與光形態發生抑制者COP1之間的橋樑。FIN219屬於GH3-like基因家族成員之一，可受生長素Auxin誘導而快速地表現，由microarray結果指出FIN219也會影響許多位於細胞核中的轉錄因子的表現，暗示FIN219可能會直接或間接調控這些轉錄因子。因此我們建構了由FIN219啟動子(promoter)接上報導基因GUS與FIN219 cDNA的轉殖株來幫助了解FIN219在植物中的表現與調控情形。結果發現轉殖在fin219-null突變株背景下，FIN219會特別表現在植株葉緣出水孔(hydathode)與附近的保衛細胞及毛茸(trichomes)基部、初生葉、下胚軸、根部等處，然而在phyA缺失的時候植株幼苗的FIN219則不會在下胚軸表現。報導中已知JAR1和FIN219是同一個基因，JAR1參與在JA的訊息傳遞中，修飾JA分子使JA接上isoleucine (Ile)，成為具有活性的狀態，使JA訊息可以傳遞到下游。而COI1 (coronatine insensitive 1)在JA訊息傳遞中扮演負調控者的角色。Microarray結果顯示在遠紅光下fin219突變會使植株中COI1的表現量大幅下降，表示FIN219的功能正常與否會影響到COI1基因的表現。在遠紅光下生長的coi1-16突變株也呈現出對遠紅光不敏感的外表型，有較野生型抽長的下胚軸，顯示COI1可能參與了遠紅光的訊息傳遞。由RT-PCR結果顯示fin219突變在遠紅光下的確會使COI1基因表現量下降，而coi1-16突變株在遠紅光下FIN219基因的表現也同樣降低，顯示COI1與FIN219彼此之間有正向調控的關係。它們彼此更深入的相互關係會在論文中討論。Light is one of the major environmental signals that influence plant growth and development. Plants can detect almost all facets of light such as direction, duration, quantity and quality with three major classes of photoreceptors: phytochromes, cryptochromes and phototropins. Jasmonic acid (JA) has been recognized as a phytohormone, which is involved in both developments and defense pathways in Arabidopsis. How the integration of light and JA affects Arabidopsis development remains to be elucidated.IN219 is a component of phyA-mediated far-red light signaling pathway. It is highly similar to a family of proteins defined by the soybean early auxin-inducible gene GH3. FIN219 expression is rapidly induced by auxin. According to the result of microarray data, FIN219 affects the expression of several transcription factors which are localized in the nucleus, suggesting that FIN219 may directly or indirectly regulate these nuclear proteins. So we produced Arabidopsis transgenic plants containing a FIN219 promoter driven GUS-FIN219 fusion construct to understand the function of FIN219. In the fin219-null mutant background, GUS staining can be detected in the hydathode of cotyledons, and the guard cells around hydathodes. GUS staining are also found in the base of trichomes, hypocotyls and roots. In contrast, in the phyA mutant background, GUS-FIN219 was not expressed in the hypocotyls. AR1 has been shown to be the same locus as FIN219 and functions as an enzyme to conjugate isoleucine to JA. So FIN219 is also involved in JA signaling pathway. Microarray data reveal that the COI1 expression is decreased dramatically in fin219 under FR condition. Here we found that coi1-16 is insensitive to far-red light. RT-PCR results indicate that COI1 expression is down-regulated in fin219-null matant and FIN219 expression also down-regulated in coi1-16 under far-red light, which implies that they positively regulate with each other. Further regulatory relationship will be discussed in the thesis.目錄 I文摘要 III文摘要 V寫對照表 VII一章 前言 1、 緒論 1、 光敏素 2、 光訊息傳遞 3、 FIN219基因 4、 COI1基因 5、 保衛細胞的調控 5、 研究目標 7二章 材料與方法 8、 載體構築 (Plasmid Construction) 8、 植物材料與生長條件 8、 基因轉殖與轉殖株篩選 9、 GUS活性染色分析 9、 RNA表現量分析 9、 蛋白質表現分析 10、 雜交測試 11三章 結果 12、 載體的構築 12、 p219:GUS轉殖株受光影響的GUS表現分析 12、 質體的轉殖與阿拉伯芥轉殖株的分析 13、 COI1突變株外表型分析 15四章 討論 18、 p219:GUS-FIN219表現在葉表皮的保衛細胞 18、 p219:GUS-FIN219並無法rescue fin219的突變外表型 19、 COI1與FIN219以及光訊息傳遞的關聯 20、 coi1突變株對光及JA的反應 21、 未來展望 21五章 結果圖片 22考文獻 44圖 55錄一、實驗操作流程 60錄二、一般藥品配製 76application/pdf3521686 bytesapplication/pdfen-US阿拉伯芥光訊息傳遞FIN219COI1Arabidopsislight signaling transductionhttp://ntur.lib.ntu.edu.tw/bitstream/246246/181941/1/ntu-98-R95b42021-1.pdf