謝旭亮臺灣大學：植物科學研究所蕭志強HSIAO, CHIH-CHIANGCHIH-CHIANGHSIAO2007-11-272018-07-062007-11-272018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/57980光對於植物來說是一項非常重要的環境因子，它會影響到植物許多方面的生長發育。阿拉伯芥在演化過程中，發展出了一套非常複雜的光訊息接受體系統，用來接受不同的光訊息。先前的研究中找到的FIN219基因，會參與在Phytochrome A (PHY-A)調控的遠紅光傳遞路徑上。而FIN219基因屬於阿拉伯芥中GH3-like基因家族的一員，這群基因家族在阿拉伯芥中共有19個成員。先前的研究中，利用RT-PCR的分析，發現在各種光源之下，GH3-14與GH3-15會受到好幾個光訊息傳遞者突變株的影響。另外，這兩個基因都屬於阿拉伯芥中GH3-like基因的第三群。因此，為了了解這兩個基因之間，以及和FIN219之間的關係，本篇研究便針對他們的突變株和轉殖株作為研究對象。 在RT-PCR的結果中，發現在各種光源之下(遠紅光、紅光、藍光、黑暗以及白光)，fin219突變株中的GH3-14表現量比野生型(Col)中有增加表現的情形，而GH3-15在fin219突變株中，以紅光和藍光處理後，反而會有下降表現的情形。這樣的結果顯示FIN219對於GH3-14與GH3-15的調控是不一樣的。 進一步利用GH3-14與GH3-15的T-DNA插入突變株為材料，發現這兩個基因的T-DNA插入突變株會造成GH3-14基因表現的增加，而GH3-15的表現下降，並且都會造成遠紅光下的幼苗比Col長的下胚軸。而在GH3-14的大量表現轉殖株中，也有跟前述相同的表現情形。另外，T-DNA插入突變株都會造成成株在長日照與短日照之下，有延遲開花的現象。這些結果都顯示了GH3-14對於遠紅光的訊息傳導與開花的調控，是一個負向調控因子，而GH3-15則是正向調控因子。 另一方面，GH3-14與GH3-15在細胞中的表現位置，以及promoter的活性分析，都可以觀察到這兩個基因都會在同樣的地方表現，表示這兩個基因有可能是藉由相互結合而去執行它們的功能。 在上位性分析的實驗中，發現FIN219在遠紅光的訊息傳遞，是位於GH3-14與GH3-15的下游，因此這三個基因之間的調控關係，是複雜且值得深入研究的。Light is one of the most important environmental cues as well as signal transduction factors for plants. Arabidopsis has evolved complex photoreceptor systems to perceive different light signals. FIN219 has been shown to be involved in phytochrome A (phyA)-mediated far-red light signaling and belongs to a GH3-like gene family that is composed of 19 members in Arabidopsis. Previous studies by RT-PCR revealed that both GH3-14 and GH3-15 were affected by multiple photoreceptors and signaling components under various light conditions. As well, both genes are very similar at the amino-acid level and belong to the group III of the GH3-like gene family. To further understand the functional relationship of these two genes, the thesis will focus on the functional characterization of them by transgenic approach. RT-PCR results indicated that GH3-14 transcripts were increased in the fin219 mutant compared to that in wild-type seedlings grown under continuous far-red light (cFR), red light (R), blue light (B), dark (D), and white light (WL) for 3 days; whereas GH3-15 transcripts were decreased in the fin219 mutant under red light and blue light compared to wild type, implying that FIN219 as a positive regulator in FR signaling can differentially regulate both GH3-14 and GH3-15 expression. In the light switch test, both the expression levels of GH3-14 and GH3-15 were increased in the fin219 mutant. Furthermore, T-DNA insertion lines of GH3-14 and GH3-15 and the overexpression lines of GH3-14 with increased expression of GH3-14 mRNA levels as well as decreased expression of GH3-15 mRNA levels exhibited a long hypocotyl phenotype under cFR; T-DNA insertion lines of both genes also exhibited delayed flowering phenotypes under long-day and short-day conditions. These results suggested that GH3-14 may be a negative regulator in the far-red light signaling and flowering; GH3-15 may be a positive regulator in these pathways. In addition, subcellular localization studies and promoter activity analysis revealed that both gene products might be located together in cells and in organs. Epistatic analyses revealed that FIN219 was located downstream the GH3-14 and GH3-15 in far-red light signal transduction pathway, implying that the relationship between FIN219 and GH3-14, -15 might be complicated and worth pursuing further.中文摘要..................................................1 英文摘要..................................................2 第一章 前言 一、緒論..................................................3 二、Phytochromes (PHYs)光敏素.............................3 三、Cryptochromes (CRYs) 隱花色素.........................6 四、Phototropins (PHOTs) 向光素...........................8 五、光訊息傳遞下游因子....................................9 六、植物荷爾蒙與光訊息傳遞之間之相關性...................12 七、GH3基因..............................................12 八、FIN219基因...........................................13 九、阿拉伯芥中GH3-like基因家族...........................14 十、GH3-like基因家族中的一員-- GH3-10 (DFL2) ............15 十一、GH3-like基因家族中的兩個成員—GH3-14和GH3-15.......16 十二、研究目標...........................................17 第二章 材料與方法 一、植物材料與生長條件...................................18 二、GH3-14與GH3-15基因construct的構築....................18 三、基因轉殖與轉殖株篩選.................................20 四、GUS活性染色分析......................................20 五、RNA的萃取與RNA表現量分析.............................21 六、次胞層次座落位置與基因槍法...........................21 七、雜交測試.............................................22 第三章 結果 一、在阿拉伯芥野生型Col與fin219突變株中，利用不同光源處理後，GH3-14與GH3-15的基因表現.............................23 二、不同天數的遠紅光與黑暗處理，對於GH3-14與GH3-15表現趨勢的影響...................................................24 三、在GH3-14與GH3-15的T-DNA插入突變株中，對此二基因表現的影響.......................................................25 四、GH3-14與GH3-15的T-DNA插入突變株對於阿拉伯芥外表型的影響.......................................................26 五、GH3-14基因大量表現的轉殖株對阿拉伯芥外表型的影響.....26 六、GH3-14和GH3-15對於阿拉伯芥開花時間的影響.............27 七、GH3-14與GH3-15主要表現於細胞質中，且不會受到光線照射而影響蛋白質在細胞中的座落位置.............................28 八、阿拉伯芥中GH3-14與GH3-15的promoter活性染色分析.......29 九、GH3-14與GH3-15在阿拉伯芥的不同組織器官的表現.........30 十、GH3-14及GH3-15與FIN219之間的遺傳關係.................30 結果圖表.................................................32 第四章 討論 一、FIN219對GH3-14基因表現的影響.........................54 二、FIN219對GH3-15基因表現的影響.........................55 三、GH3-14與GH3-15基因表現在fin219中會隨著生長時間的增加而減少表現.................................................56 四、GH3-14和GH3-15具有互相抑制表現的關係.................56 五、GH3-14與GH3-15之間具有非等位非互補 (nonallelic noncomplementation)的關係................................58 六、GH3-14與GH3-15在細胞、組織及器官層次上有相同的表現位置.......................................................58 七、GH3-14與GH3-15會影響阿拉伯芥下胚軸的外表型以及開花時間.......................................................60 八、GH3-14與GH3-15對於阿拉伯芥的色素累積有某種程度的影響.......................................................61 九、結論：GH3-14、GH3-15與FIN219之間的相關性，以及它們對於阿拉伯芥外表型的影響.....................................62 十、未來展望.............................................62 參考文獻.................................................64 附圖一、三種主要的光訊息接受體...........................79 附圖二、Phytochrome基因家族的五個成員....................80 附圖三、光訊息傳遞路徑...................................81 附圖四、阿拉伯芥中的GH3-like基因家族成員.................82 附圖五、GH3-14與GH3-15的表現量在不同光源下，受到不同光訊息傳遞者的影響.............................................83 附圖六、GH3-like基因家族會受到不同荷爾蒙所調控...........84 附圖七、阿拉伯芥中的GH3-like基因家族，胺基酸序列的相似性程度比較...................................................85 附圖八、實驗中所使用的載體...............................86 附圖九、GH3-14與GH3-15的cDNA序列比對.....................87 附圖十、GH3-14與GH3-15的蛋白質序列比對...................90 附錄一、一般藥品之配製...................................91 附錄二、詳細實驗方法與步驟...............................933118574 bytesapplication/pdfen-US阿拉伯芥光敏素遠紅光GH3ArabidopsisPhytochrome,far-redotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/57980/1/ntu-95-R93b42004-1.pdf