阿拉伯芥AtMAPRs之組織及細胞內定位探討

Abstract

我們在阿拉伯芥中發現四個豬的MAPR (putative membrane-associated progesterone receptor component 1) 同源蛋白質，分別為AtMAPR2、AtMAPR3、AtMAPR4 以及AtMAPR5。在先前的研究中指出，位於細胞膜上之AtMAPR5 (putative membrane steroid binding protein, MSBP1) 可在胞外與黃體素及芸苔素內酯結合，且黑暗培養可抑制在下胚軸中該基因之表現，受到光照則可回復表現，推測在阿拉伯芥中功能為細胞延長之負調控因子。AtMAPRs家族蛋白質中其他三者的功能則尚未明瞭，因此嘗試以細胞、組織定位及觀察過量表現AtMAPRs基因轉殖株之性狀，探討AtMAPRs可能之生理意義。在本論文中利用基因槍將AtMAPR2與GFP之融合基因轉殖於洋蔥表皮細胞，由GFP螢光位置以得到AtMAPR2在細胞中之定位，結果發現AtMAPR2雖然有較為聚集在細胞核內之情形，但也散佈於細胞質中；而使用GUS報導基因的暫時性表現結果也間接說明AtMAPR2並非核蛋白。另一方面，利用農桿菌轉殖法得到過量表現AtMAPR2-GUS-GFP之阿拉伯芥T2轉殖株，使用共軛焦顯微鏡觀察其幼苗之根部及下胚軸的螢光表現，發現AtMAPR2會聚集在細胞中的某種胞器內且並非細胞核。而藉由GUS染色發現，經過不同光照處理培養之T2轉殖株中，AtMAPR2在下胚軸處也許可藉由全日光照而增加其蛋白質之穩定性，且可能在植株發育的前四天即決定AtMAPR2之穩定性，之後便無法以光照或黑暗對其穩定與否產生影響。至於藉由不同賀爾蒙處理下，過量表現AtMAPR2轉殖株根部的發育，推測其直接或間接參與了由auxin所調控之根部延長。這些結果不但有助於釐清AtMAPRs家族之可能生理意義，未來更可將過量表現AtMAPR2之轉殖株與RNAi或T-DNA突變株進行雜交，以期能藉由觀測性狀改變而確定其可能之參與之訊息傳遞路徑。

Four porcine MAPR (putative membrane-associated progesterone receptor component 1) homologs in Arabidopsis were named as AtMAPR2, AtMAPR3, AtMAPR4, and AtMAPR5. In recent study, it is demonstrated that AtMAPR5 (also defined as putative membrane steroid binding protein, MSBP1) localizes to plasma membrane and binds to progesterone and brassinolide in vivo. The expression of AtMAPR5 in hypocotyls is inhibited in the dark, and is activated under continuous illumination. It suggests that AtMAPR5 is a negative regulator of cell elongation. However, the functions of other homologs are still unclear. In this study, we tried to find the possible functions of AtMAPRs by the tissue or subcellular distribution of GFP or GUS-fusion protein expressed in transgenic plants followed by phenotype analysis. Using particle bombardment, the transient expression of AtMAPR2-GFP fusion protein in onion epidermal cells showed that although in some cases AtMAPR2 aggregated in the nucleus, the cytoplasm also contained the green fluorescence. Besides, the transient expression of AtMAPR2-GUS in onion cells suggested that AtMAPR2 is not a kind of nuclear protein. We established a transgenic T2 plant overexpressing AtMAPR2-GUS-GFP by Agrobacterium-mediated transformation. It is interesting that in the roots and hypocotyls of transgenic seedlings the AtMAPR2-GUS-GFP was located in some organelles but may not be in nucleus as seen under confocal laser microscope. The GUS staining analysis of transgenic T2 seedlings grown under different light conditions demonstrated that light might increase the stability of AtMAPR2 in hypocotyls during the first four days following germination. And the stability would not be abolished or complemented by the darkness or light for the 4-d-old seedlings. The possible function of AtMAPR2 could be predicted by observing the roots of transgenic plants grown on vertical medium containing different hormones. AtMAPR2 might be involved in the root elongation regulated by auxin. These results can help us to understand the possible roles of AtMAPRs in the signal transduction. The overexpressing-AtMAPR2 transgenic T2 plants can be used to cross with RNAi or T-DNA mutants in the future and help us to further identify its function.