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Title: | 在阿拉伯芥中興FIN219交互作用蛋白質之功能分析 Functional Studies of FIN219-Interacting Proteins in Arabidpsis |
Authors: | I-Ching Huang 黃怡靜 |
Advisor: | 謝旭亮 |
Keyword: | NULL |
Publication Year : | 2004 |
Degree: | 碩士 |
Abstract: | 為了更瞭解阿拉伯芥生長發育過程裡,FIN219在光訊息傳遞中所扮演的角色,利用酵母菌雙雜交法(yeast two-hybrid)找出在阿拉伯芥cDNA library中跟FIN219有交互作用之蛋白質。我們找出一個基因,它的產物可跟FIN219的C端有交互作用,並命名為FIPl(FIN219-interacting protein 1),此基因轉譯出一個24 kD的GST(2,4-D induced glutathione S-transferase,Atlg78370);另一個基因產物跟FIN219的C端也有交互作用,命名為FIP2(FIN219-interacting protein 2),此基因轉譯出一個79 kD的GluRS (glutamyl-tRNA synthetase,At5g26707)。本篇論文將FIP2大量表現在Columbia野生型阿拉伯芥和fin219突變體,觀察它可能的外表型;結果顯示,FIP2的大量表現體在遠紅光下,其下胚軸比野生型阿拉伯芥長,但較加fin219突變體短。另外,亦發現FIP2 T-DNA插入的轉殖株,在紅光、遠紅光中,其下胚軸比野生型阿拉伯芥長,但較加fin219突變體的短,其中調控的關係目前還不清楚。本篇論文主要是針對FIP1作較詳盡的研究。 FIP1 cDNA有654 bp,轉譯出一個217氨基酸、24 kD的蛋白質。Northern分析的結果顯示,此基因是可以受到auxin及jasmonic acid的誘導。此外,當FIP1大量表現在Columbia野生型阿拉伯芥時,發現轉植株的下胚軸在連續遠紅光或紅光照射下表現出不敏感(hyposensitive)的外表型,即較野生型(WT)阿拉伯芥長的下胚軸;而大量表現在加fin219突變體中時,則無任何外表型,暗示它的表現與有正確功能的FIN219有關。另外,FIP1 antisense轉殖株的外表型則呈現比野生型阿拉伯芥短的下胚軸,由此推測FIP1在遠紅光所調控的抑制下胚軸的途徑裡扮演一負向調節者。由北方墨漬法分析亦顯示,FIP1基因在照射白光、遠紅光中表現量有增加的趨勢;但是在藍光、紅光和黑暗中FIP1的表現並不明顯。此外,亦發現在光敏素A (phytochromo A)、光敏素B和其他光訊息傳遞途徑的突變體中,例如fin219和fhy,FIP1 mRNA的表現量下降。 而基因的表現,也與蛋白質在細胞內的位置有關;利用基因槍方式將GUS-FIP1質體送到洋蔥表皮細胞,發現GUS-FIP1融合蛋白表現在細胞質和細胞核,而且此細胞內的位置,並未受光的影響而改變。綜合目前研究顯示,FIP1和FIN219有交互作用且參與光訊息傳遞,特別是遠紅光和紅光。 To further understand the function of FIN219 in light signaling during Arabidopsis development, a yeast two-hybrid approach was utilized to isolate FIN219-interacting partners. A gene FIP1 (FIN219-interacting protein 1) encoding a 24 kD of GST(2,4-Dinduced glutathione S-transferase,At1g78370) was recovered and interacting with the C-terminus of FIN219. Another FIN219-interacting partner FIP2 (FIN219-interacting protein 2) encoding a 79 kD of G1uRS (glutamyl-tRNA synthetase,at5g26707) was recovered and interacting with the C-terminus of FIN219, too. The transgenic plants overexpressing the FIP2 gene in wild type Columbia display a hyposensitive phenotype with longer hypocotyls than wild type in far red (FR) light. However, FIP2 T-DNA insertional mutants also exhibit a longer hypocotyl phenotype in both FR and R light conditions. The reasons that two opposite constructs generate similar results remain to be determined. So the thesis will focus on the characterization of FIP1 gene functions in Arabidopsis. FIP1 gene encoding a 24 kD of protein with 217 amino acids can be induced by auxin and jasmonic acid according to the results of Northern analysis. The transgenic plants overexpressing the FIP1 gene in wild type Columbia display a hyposensitive phenotype with longer hypocotyls under continuous far-red and red light and this longer hypocotyl phenotype is regulated by FIN219; however, FIP1 antisense lines show an opposite phenotype, suggesting that FIP1 may act as a negative regulator for FR-mediated inhibition of hypocotyl elongation. Furthermore, the results of Northern blot analyses indicated that FIP1 gene expression is upregulated by white light and detectable in FR, but its expression is hardly detected in dark ,blue and red light conditions. Besides, its expression is reduced in phytochrome A and B mutants and several light signaling transducer such as fin2l9 and fhy1 mutants. The GUS-FTP1 fusion protein is localized in both nucleus and cytoplasm based on the result of transient assays in onion cells. Taking all current results together, the FTP1 is interacting with F1N219 and involved in light signaling pathway, especially in FR and R light spectrum. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24165 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 植物科學研究所 |
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