https://scholars.lib.ntu.edu.tw/handle/123456789/139431
標題: | 阿拉伯芥中FIN219基因表現的功能性研究 Functional studies of FIN219 gene expression in Arabidopsis |
作者: | 曾美瑄 Tseng, Mei-Hsuan |
關鍵字: | 阿拉伯芥遠紅光訊息傳遞;phytochrome A | 公開日期: | 2004 | 摘要: | 植物在照射光之後,分別由不同的光接受體接受光的刺激,光接受體在接受光的訊息之後,藉由位於下游的訊息傳遞分子傳送光訊息,影響植物體的生長發育。在遠紅光照射下,阿拉伯芥的fin219突變體幼苗具有下胚軸伸長的外表型,利用基因定位(map-based method)的方法,已將FIN219基因分離。FIN219屬於GH3基因家族的成員,由575個胺基酸組成,分子量大小約為64kD;在其N端及C端各有一個coiled-coil區域,可能與蛋白質間的交互作用有關。然而目前對於FIN219的功能以及其作用機制尚不明確,有待進一步的研究。 利用大量表現FIN219及轉入FIN219 N端反譯股的轉殖株,來研究FIN219基因對於植物生長發育的影響。在大量表現FIN219的轉殖株中,我們篩選到遠紅光下兩種不同的外表型,一種對於遠紅光有高度敏感的外表型,其下胚軸長度比野生型短;另一種對於遠紅光不敏感,其下胚軸長度比野生型長。利用北方式點墨法分析其FIN219基因表現量,發現白光下在較短下胚軸的轉殖株中表現量比野生型多,而較長下胚軸的表現量則與野生型無差異。在蛋白質層次上利用西方式點墨法分析蛋白質表現,發現無論是轉殖株或者是野生型其FIN219蛋白質的表現量皆無明顯差異。而在FIN219 N端反譯股的轉殖株中,在遠紅光下其下胚軸長度介於野生型及fin219突變體之間,分析RNA的表現量也發現其在部分株系當中表現量有比野生型下降的情形,蛋白質含量與野生型相比無明顯差異。由此兩種轉殖株實驗分析可以發現FIN219基因在遠紅光抑制下胚軸延長的生理機制上扮演正調控的角色。另外在外表型的觀察上也發現大量表現FIN219基因時會造成植物部分花朵不稔,其花瓣沒有對稱性,大小也不一,雌蕊呈現螺旋狀。當FIN219基因表現量下降時則會使植物葉子呈現黃綠色,植株矮小,且開花期延後,但是FIN219基因是如何影響這些生理機制還不明確。 另外針對FIN219蛋白質的功能,利用西方式點墨法,研究FIN219在不同光源與光訊息傳遞有關的突變體中的表現。發現FIN219會受到遠紅光誘導表現增加;cop1不同等位基因突變體在黑暗中其FIN219具有差異性表現,另外在藍光接受體cry1及cry2的突變體中FIN219表現量下降,而大量表現FIN219基因的轉殖株在藍光下也有對藍光高度敏感的外表型,認為FIN219蛋白質可能也參與藍光的訊息傳遞鏈。 利用PLACE軟體分析發現,在FIN219啟動子上有多個受到低溫誘導的功能區存在,但是利用RT-PCR檢驗植物處理不同時間低溫的FIN219基因表現時,發現表現量會迅速的降低,然後再漸漸的增加至低溫處理後16小時,之後再降低,但蛋白質沒有明顯改變。在研究FIN219蛋白質是否具有組織專一性時,發現在根部無法偵測到蛋白質表現,且在突變體葉子的表現量比野生型來的少。此外我們發現FIN219蛋白質在某些光源或者突變體當中,會出現兩條大小相差約8~10kD的條帶。在加入去磷酸根酵素的實驗中發現,FIN219蛋白質的分子量大小會有改變。推測FIN219蛋白質可能具有兩種型態,會專一的在特定的組織部位或者是細胞中表現,在不同生長時期進行兩種型態間的轉換,以調控植物的生長發育。 After light irradiation, Plants are able to sense the changes of light environments by different photoreceptors, and then transduce the signal via light signaling components to regulate plant growth and development. fin219 mutant displays a long hypocotyl phenotype under continuous far red(cFR) light. The FIN219 gene has been isolated by map-based method and encodes a protein with 575 amino acids, which belongs to a GH3-like gene family in Arabidopsis. Furthermore, two coiled-coil domains, believed to be involved in protein-protein interaction, are found in FIN219 with one in the N-terminus and the other in the C-terminus. Currently, however, FIN219 function and regulatory mechanism are not clear yet and remain to be solved. Transgenic plants overexpressing FIN219 or antisensing the N-terminus of FIN219 was used to study the effect of FIN219 on plant growth and development. Two different phenotypes of transgenic plants overexpressing the FIN219 grown in cFR were obtained, one with hypersensitive, shorter hypocotyl phenotype and the other with hyposensitive, longer hypocotyl phenotype than wild type. FIN219 expression in the shorter hypocotyls of transgenic plants grown in white light was investigated by Northern blot and found that it was indeed expressed more than that in wild type. However, its expression in the longer hypocotyls of transgenic plants didn’t show significant difference from that in wild type. Further, FIN219 protein expression in transgenic plants didn’t show significant difference when compared with wild type by Western blot analysis. In addition, transgenic plants antisensing the N-terminus of FIN219 exhibited intermediate hypocotyls in cFR compared to wild type and fin219 mutant. FIN219 mRNA level assayed by Northern was also reduced in some of the transgenic lines, but its protein level didn’t show obvious difference from wild type. Based on the results of these transgenic studies, it indicated that FIN219 acts as a positive regulator to transduce FR-mediated inhibition of hypocotyl elongation. Besides, the transgenic plants overexpressing FIN219 display a partial sterile flower phenotype, unequal size of asymmetric petals and spiral carpals. The antisense transgenic plants with lower level of FIN219 expression did show yellowish color of leaves, dwarf phenotype and a delayed flowering. That how the FIN219 affects all these phenotypes remains unclear. To further understand regulatory mechanisms of FIN219 expression in light signaling, we investigate its expression in different light conditions and photomorphogenic mutants by western blotting. The results indicated that FIN219 protein was induced by cFR. FIN219 was also shown with different expression patterns in different cop1 alleles under dark and white light conditions. Furthermore, FIN219 protein expression was reduced in cry1 and cry2 mutants, and transgenic plants with FIN219 overexprssion also have a hypersensitive phenotype under blue light, implying that FIN219 may be involved in blue light signaling as well. The result of promoter analysis by PLACE program indicated that there are several cold-activated cis-elements present in the promoter of FIN219 gene. Consistent with this, the result from cold-induced experiment showed that FIN219 mRNA expression detected by RT-PCR was reduced rapidly after cold treatment, then gradually increased up to 16 hours after cold treatment and then dropped again. However, FIN219 protein level did not change too much. In addition, FIN219 displays a tissue-specific manner with down-regulated in roots and reduced in the leaves of fin219 mutants compared to that in wild type. Interestingly, FIN219 exhibits two close sizes of bands with about 8 kD difference when detected by specific antibodies raised against the N-terminus of FIN219. The lower band will be shifted to the upper one after treated with phosphatase, suggesting that FIN219 protein may exist two forms to regulate plant growth and development. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/57976 | 其他識別: | zh-TW |
顯示於: | 植物科學研究所 |
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ntu-93-R91226019-1.pdf | 23.31 kB | Adobe PDF | 檢視/開啟 |
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