水稻 ATP-Binding Cassette (ABC) 轉運蛋白 OsABCG36 的功能性分析

Abstract

ABC 轉運蛋白 (ATP-Binding Cassette transporter) 為一廣泛存在於各物種的龐大膜轉運蛋白家族，在多種基質的運輸上扮演重要角色，包括胺基酸、多肽鏈、脂質及金屬離子等。水稻具有超過130個 ABC 轉運蛋白，但對其功能了解非常少。我們過去的研究發現，屬於水稻PDR類的ABC轉運蛋白基因OsABCG36，其表現受到多種重金屬高度誘導。為瞭解其在水稻中扮演的角色，本研究進一步針對OsABCG36進行功能性分析。酵母菌功能性分析結果顯示，大量表現 OsABCG36 會專一性的促進鈷顯著累積而對酵母菌造成毒害。水稻功能性分析顯示，OsABCG36-RNAi基因靜默水稻對於鈷具有較高的敏感性；相反地，OsABCG36-OE 大量表現水稻具有較高的鈷耐受性。鈷累積量分析發現不論OsABCG36-RNAi及OsABCG36-OE都與野生型無明顯差異。次細胞定位結果顯示，OsABCG36-GFP融合蛋白可能位於葉綠體與細胞膜上。基因表現分析結果顯示，OsABCG36 基因表現會隨著鈷處理濃度和時間而受到大幅誘導，其中梗稻TNG67和秈稻TN1根部以2.5 mM氯化鈷處理，可分別誘導856倍及2536倍，在地上部則分別誘導22倍及9.8倍。啟動子特性分析顯示，POsABCG36(1K)/GUS、POsABCG36(1.5K)/GUS以及POsABCG36(2K)/GUS轉殖水稻在葉、葉環、葉鞘及節皆有GUS表現，並受鈷誘導。分析隱子 (intron) 對OsABCG36啟動子的活性影響發現，第一個隱子可增加重金屬誘導性，第二個隱子可增強啟動子在葉部活性，第三個隱子則抑制啟動子活性。以鈷處理水稻幼苗發現，高濃度的鈷會造成水稻的毒害，並抑制水稻幼苗根部的發育。以上結果顯示，OsABCG36是一個鈷轉運蛋白，在酵母菌中可能為一個鈷吸收轉運蛋白，在植物中，OsABCG36可能位於葉綠體與細胞膜上，並影響細胞內的鈷分布

ATP-binding cassette (ABC) transporters is a membrane transporter superfamily which ubiquitously existed in all living organisms, and play important roles in transporting various substances, including amino acids, polypeptides, lipids and metal ions. Rice has more than 130 ABC transporters, but little is known about their functions. Our previous study indicated that the rice PDR-type ABC transporter OsABCG36 was highly induced by various heavy metals. To dissect the function of OsABCG36, yeast and rice were used to evaluate the transport of metals. Overexpression of OsABCG36 increased cobalt (Co) accumulation and caused toxic effect to yeast. In rice, OsABCG36-RNAi transgenic rice showed a Co sensitive phenotype. Conversely, OsABCG36-OE transgenic rice showed a Co tolerant phenotype. However, the Co concentration of OsABCG36-RNAi and OsABCG36-OE was similar as compared to wild type. Subcellular localization analysis showed that OsABCG36 localized at chloroplast and plasma membrane. The analysis of OsABCG36 expression indicated the transcript level of OsABCG36 was highly induced in both japonica and indica rice, moreover, it was increased along with the increase of Co concentration and time. In addition, treatment of japonica and indica rice with 2.5 mM CoCl2, the transcript of OsABCG36 in root was increased 856 folds and 2536 folds, and that in shoot was also increased 22 folds and 9.8 folds, respectively. According to the analysis of promoter characterization, GUS was induced by Co and that accumulated in blade, collar, sheath, and node of POsABCG36(1K)/GUS, POsABCG36(1.5K)/GUS and POsABCG36(2K)/GUS transgenic rice. The activity of OsABCG36 promoter was also affected by itself introns. The first intron of OsABCG36 could enhance the inducibility of OsABCG36 promoter to heavy metals; the second one could enhance the OsABCG36 promoter activity in shoot; the third one inhibited the promoter activity. To figure out the physiological role of Co in rice seedlings, the treatment of various Co concentrations was conducted. Higher Co concentrations (> 50 uM) could result in Co toxicity in rice seedling, and also inhibit the root growth. Eventually, OsABCG36 is a Co transporter, and might have influx function in yeast. In plants, OsABCG36 might localize at chloroplast and plasma membrane, and function as cellular Co distribution.