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National Chung Hsing University Institutional Repository - NCHUIR > 農業暨自然資源學院 > 生物科技學研究所 > 依資料類型分類 > 碩博士論文 >  阿拉伯芥中兩個Forever Young Flower (FYF) 下游基因之功能性分析

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152586

標題: 阿拉伯芥中兩個Forever Young Flower (FYF) 下游基因之功能性分析
Characterization and Functional Analysis of Two Forever Young Flower (FYF) Downstream Genes in Arabidopsis thaliana
作者: 李孟珊
Li, Meng-Shan
Contributors: 楊長賢
Chang-Hsien Yang
生物科技學研究所
關鍵字: IAA33;C2H2-ZFP;FYF下游基因
IAA33;C2H2-ZFP:FYF downstream genes
日期: 2013
Issue Date: 2013-11-18 10:44:07 (UTC+8)
Publisher: 生物科技學研究所
摘要: 在阿拉伯芥中異位表現MADX box基因-Forever Young Flower(FYF)會延遲花器的老化與脫離。藉由microarray的分析,發現到IAA33為受到FYF所抑制的下游基因。IAA33是AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA)這群基因的其中一員。在IAA33::GUS轉基因植物中,GUS蛋白的活性表現在根尖、七天和十四天大小苗的葉子、年輕花苞的花萼和成熟花的雄蕊和雌蕊。為了進一步探討IAA33這個基因的功能,將所選殖出的IAA33基因之序列構築於大量表現(35S啟動子)、強烈抑制(SRDX)及強烈活化(VP16)之載體中。研究結果顯示,35S::IAA33與35S::IAA33-SRDX轉殖株皆有觀察到提早開花、葉子捲曲和花器有提早老化的現象,而35S::IAA33-VP16轉殖株則與野生型阿拉伯芥相似。此外,我們將FYF和IAA33共同大量表現至阿拉伯芥。發現在大量表現FYF 所造成的延緩花器老化及脫落的現象,會因為共同大量表現IAA33而消失,結果顯示IAA33為FYF之下游基因。由以上結果顯示,IAA33為轉錄抑制子且會被FYF所抑制進而調控花器的老化。除了IAA33之外,At5g15480(C2H2-ZFP)也被發現為受FYF所調控的基因之一,在C2H2-ZFP::GUS轉基因植物中,GUS蛋白的活性表現在七天和十四天大小苗的葉子以及根、年輕花苞的花萼和成熟花的花蕚。為進一步探討C2H2-ZFP這個基因的功能,將所選殖出的C2H2-ZFP基因之序列構築於大量表現(35Sp)、強烈抑制(SEDX)及強烈活化(VP16)之載體中。研究結果顯示,35S:: C2H2-ZFP、35S:: C2H2-ZFP-SRDX與35S:: C2H2-ZFP-VP16轉殖株皆觀察到花器有較延緩老化以及花藥不完全開裂的現象。藉由研究分析顯示C2H2-ZFP所具有延緩花器老化及脫落之性狀,可能是由於乙烯訊息傳導路徑下游基因EDF1-4被抑制所造成的。而花藥開裂不完全可能也是由於乙烯訊息傳遞受到抑制所導致。
Ectopic expression of Forever Young Flower (FYF), a MADS box gene of Arabidopsis, caused significantly delayed flower senescence and abscission in transgenic Arabidopsis. In microarray analysis, one gene IAA33 was found down-regulated in 35S::FYF transgenic plants. IAA33 is a member of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family. In IAA33::GUS Arabidopsis, the GUS activity were specifically detected in root tips, young emerged leaves of 7- and 14-day-old seedlings, sepals of flower buds, pollen and ovules of mature flowers. To determine the physiological function of IAA33, we performed transgenic analysis in Arabidopsis by overexpression (35S promoter), repression (fusion of SRDX) and activation (fusion of VP16) of IAA33. The result indicated that 35S::IAA33 and 35S::IAA33-SRDX caused similar abnormal phenotypes such as early flowering, leaf incurvature and the promotion of flower senescence whereas the phenotypes of 35S::IAA33-VP16 looks like wild type plants. Furthermore, ectopic expression of IAA33 in 35S::FYF plants promoted flower senescence and abscission. This result confirmed that IAA33 is a downstream gene for FYF. Our result suggested that IAA33 functions as a repressor and was suppressed by FYF in regulating flower senescence. In addition to IAA33, a gene At5g15480 (C2H2-ZFP) regulated by FYF was also identified. In C2H2-ZFP::GUS Arabidopsis, GUS activity was specifically detected in root, young emerged leaves of 7- and 14-day-old seedlings, sepals of flower buds and mature flowers. To determine the physiological function of C2H2-ZFP, transgenic Arabidopsis overexpression (35S promoter), repression (fusion of SRDX) and activation (fusion of VP16) of C2H2-ZFP were generated. 35S::C2H2-ZFP, 35S::C2H2-ZFP-SRDX and 35S::C2H2-ZFP-VP16 caused similar abnormal phenotypes such as the delay of flower senescence and incomplete defect in anther dehiscence. Further analysis indicated that C2H2-ZFP delay flower senescence by down-regulating the ethylene signaling downstream genes EDF1-4. The incomplete anther dehiscence may be also caused by the inhibition of ethylene signaling.
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