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研究生(外文):Hui-Min Chiang
論文名稱(外文):Functional Studies of FIN219-interacting Protein 2 (FIP2) in Arabidopsis Light Signaling Pathways
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對固著生長的植物來說,能夠因應外界光線環境改變,進而調整自身的生長發育是非常重要;研究植物中的光訊息傳遞,可幫助我們了解光如何影響植物的生長發育。在阿拉伯芥中,已知FIN219在phyA所調控的遠紅光訊息傳遞下扮演正向調控者角色,然而FIN219在遠紅光中作用的分子機制尚未明瞭;前人利用酵母菌雙雜交法(yeast two-hybrid)找出會與FIN219 C端交互作用的蛋白質,命名為FIP2 (FIN219-interacting protein 2),此基因即為glutamyl-tRNA synthetase (GluRS,At5g26710)。FIP2如何與FIN219交互作用,及FIP2是否有參與光訊息傳遞為本研究之目標。
從酵母菌雙雜交法及雙分子螢光互補系統的結果得知,FIP2藉由N端和FIN219 C端在細胞質中進行交互作用;且fin219 fip2雙突變體在弱遠紅光下的下胚軸的長度較fin219突變體及fip2突變體來的長,推測兩者在遠紅光下有非等位非互補(nonallelic noncomplementation )的遺傳調控關係。另外,fip2突變體、FIP2基因表現量下降的FIP2OE轉殖株#2-1-5和#2-5-6及fin219 fip2雙突變體,在遠紅光亦呈現較野生型長的下胚軸,且會影響到光訊息相關基因的表現量(例如:CHS),也會影響phyA所調控的花青素的累積及葉綠素生合成,由此可知FIP2確實參與遠紅光的訊息傳遞。
另外,FIP2基因表現量下降的FIP2OE轉殖株#2-1-5和#2-5-6及fin219 fip2雙突變體,在ABA所調控的抑制種子發芽以及根的延長均不敏感,暗示著也許FIP2也參與ABA所調控的一些生理性狀。綜合上述結果顯示FIP2參與遠紅光的訊息傳遞,會影響到花青素及葉綠素累積,也與ABA抑制種子的發芽以及根的延長有關。
For immobile plants, it is important to adjust their development in response to the changes of light environments. Through studying light signaling pathway, we can understand how light affects plant development. Far-red insensitive 219 (FIN219) has been shown to be a positive signal component in phyA-mediated FR signaling pathway, but the molecular mechanisms remain unknown. To further understand the function of FIN219 in light signaling during Arabidopsis development, a yeast two-hybrid approach has been used to isolated FIN219-interacting partners. A gene FIP2 (FIN219-interacting protein 2) encoding a glutamyl- tRNA synthetase (GluRS) was obtained and interacting with the C-terminal domain of FIN219. However, the regulation between FIP2 and FIN219 in light signaling pathway remains to be elucidated.
Furthe confirmation of FIN219 and FIP2 interaction using yeast two-hybrid and bimolecular fluorescence complementation (BiFC) methods indicaded that the N-terminal domain of FIP2 can interact with the C-terminal domain of FIN219. This interaction occurs in the cytoplasm. Furthermore, the fin219 fip2 double mutant exhibited a longer hypocotyl phenotype than its respective parental lines specifically under weak FR light. These data suggest that FIP2 and FIN219 act as a nonallelic noncomplementary genetics relation.
RT-PCR results indicated that the expression levels of light responsive genes such as CHS and RBCS were down-regulated in the fip2 mutant and transgenic seedlings with reduced levels of FIP2 under far-red light, which was consistent with the hyposensitive phenotype of the fip2 mutant as well as the transgenic seedlings with less FIP2 transcript under far-red light. Moreover, transgenic seedlings containing a reduction of FIP2 level resulted in a decrease of anthocyanin accumulation in FR, and led to a defect in the phyA regulated FR blockage of greening and a long hypocotyl phenotype compared to that of wild type under FR.These results implie that FIP2 participates in the control of hypocotyl elongation in FR-mediated light signaling. Besides, the fip2 mutant, fin219fip2 double mutant and transgenic seedlings with reduced FIP2 transcripts were less sensitive to ABA-mediated inhibition of seed germination and root elongation. Taken together, these data indicate that FIP2 may participate in FR-mediated light signaling leading to anthocyanin accumulation and FR-mediated blockage of greening, and ABA-mediated inhibition of seed germination and root elongation.
摘要 V
Abstract V
縮寫對照表 VIII
前言 1
一、 緒論 1
二、 光敏素 2
三、 植物的光訊息傳遞 2
四、 FIN219基因參與植物生長發育的調控機制 3
五、 FIP2基因 4
六、 離層酸(ABA)參與植物生長發育調控的分子機制 5
七、 茉莉酸(JA)訊息傳遞調控植物生長發育 5
八、 研究目標 6
材料與方法 7
一、 植物材料與生長條件 7
二、 Genomic DNA 萃取 7
三、 分析T-DNA homozygous lines 8
四、 雙突變株的建立與基因型判定 8
五、 RNA表現量分析 8
六、 花青素與葉綠素含量的檢測 9
七、 種子發芽測試 9
結果 12
一、 FIP2與FIN219間的交互作用 12
二、 fip2突變體 12
三、 大量表現FIP2的轉殖株 14
四、 fin219 fip2雙突變體 16
五、 FIP2參與ABA調控的生理性狀 15
六、 FIP2參與JA調控的生理性狀 16
討論 19
一、 FIP2與FIN219的交互作用位於細胞質 19
二、 FIP2參與在遠紅光訊息傳遞中 19
三、 FIP2影響ABA所調控的植物生理 21
四、 FIP2也許參與在JA所調控的植物生理 22
結論 23
結果圖片 24
參考文獻 45
附錄一、實驗操作流程 55
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