阿拉伯芥中 AtMAPR3 表現受 d-amino levulinic acid 及逆境誘導之研究

Abstract

豬的 MAPR (membrane associated progesterone receptor component 1) 與黃體激素及血基質結合有關，從阿拉伯芥中發現四個與 MAPR 相似度為30-40 % 之同源蛋白質，將其命名為 AtMAPR2、AtMAPR3、AtMAPR4 與 AtMAPR5。這四種蛋白質皆具有高度保守性的 cytochrome b5 like heme/steroid binding domain。(高艾玲，2010) 本論文針對 AtMAPR3 與血基質結合特性，探討 AtMAPR3 的表現量是否會受到血基質影響。以血基質合成前驅物 δ-aminolevulinic acid (ALA) 處理11天大阿拉伯芥幼苗，發現在正常光照下 AtMAPR3 表現量上升 3.5倍，完全黑暗下基因表現也上升2.5倍，而其他 AtMAPRs 卻不受影響。處理 ALA 後再加入 dipyridyl (DPD) 來抑制 Fe-chelatase 使得血基質無法合成，AtMAPR3 表現量上升情形即消失。AtMAPR3 可能與過氧化物的代謝有關，分別以 H2O2 及 H2O2 類似物 tert-butyl hydroxide (tBHP) 處理11天大幼苗後，AtMAPR3 表現量約上升6倍及4倍。先前文獻指出，植物對於 jasmonic acid (JA) 產生的反應可能是利用 H2O2 作為訊息分子啟動一些防禦基因之表現，以不同濃度 JA 處理 (50-100 μM)，會使 AtMAPR3 表現量上升4倍。實驗室已有 AtMAPR3 基因剔除突變株 (AtMAPR3-KO)，需要建構 AtMAPR3 基因過量表現突變株 (AtMAPR3-OX)，以瞭解 AtMAPR3 所參與的生理功能。將此兩種突變株及 WT 進行 H2O2、tBHP 及 methyl viologen (MeV) 氧化逆境處理，發現 AtMAPR-KO 存活率比 WT 略低，但無顯著差異，此外也無觀察到 AtMAPR-OX 有存活率較高現象。綜合這些實驗結果推論，阿拉伯芥細胞中 ALA 下游產物量，如血基質或 Mg-protoporphyrin IX，可能是影響 AtMAPR3 表現量的訊息分子之一。而AtMAPR3 在 ROS 所引發反應中扮演何種生理角色，需要進一步實驗探討。

Four functional-unknown proteins, AtMAPR2, AtMAPR3, AtMAPR4 and AtMAPR5, possessing sequence 30-40% similarity to MAPR (membrane associated progesterone receptor) are studied in this group. The cytochrome b5 like heme/steroid binding domain are highly conserved in these proteins, and they bind progesterone and heme in animals. The UV-visible absorption spectra showed AtMAPRs are hemoproteins. (Kao, 2010, unpublished) To study the physiological role of heme-binding ability of AtMAPR3, we analyzed whether or not the expression of AtMAPR3 are subjected to the heme concentration. In this study, 10-day-old seedlings were treated with a precursor of heme, δ-aminolevulinic acid (ALA). The results showed AtMAPR3 expression was three-fold-increased by ALA treatment under 16 h light / 8 h dark, and two-fold-increased under 24 h dark; however, when adding dipyridyl (DPD), the inhibitor of Fe-chelatase, the induction was blocked. The expression of AtMAPR3 was also induced by H2O2 and tert-butyl hydroxide (tBHP) about six-fold. H2O2 could be used as a second messenger to express the defence-related genes by jasmonic acid (JA), and JA increased AtMAPR3 expression about four-fold. To further study the physiology roles of AtMAPR3, we need to analyze the phenotype of AtMAPR3-KO and other mutants. In this study, AtMAPR3-KO, AtMAPR3-OX and WT were treated with H2O2, tBHP and methyl viologen (MeV), and the survival rate of AtMAPR3-KO was lower than WT slightly. However, AtMAPR3-OX showed no differences with WT. The results revealed that the downstream products of ALA, such as Mg-protoporphyrin IX or heme, could be a signal molecule which involved in the expression of AtMAPR3. The expression of AtMAPR3 in response to ROS needs to be further investigated in physiological aspect.