水稻谷胱甘肽還原酶2 (OsGR2) 基因功能分析

Abstract

Glutathione reductases (GR)為植物重要的抗氧化酵素，負責將氧化態的 GSSG 還原為 GSH 。水稻 GR 由三個基因組成，分別為位於細胞質的 OsGR2 ，和位於粒線體與葉綠體的 OsGR1 及 OsGR3 。利用半定量 RT-PCR 偵測 OsGR2 的基因表現情形。結果發現 OsGR2 在三葉齡幼苗的地上部及根部皆有表現，且以根部的表現量較高，而在水稻孕穗時期主要表現在劍葉、葉身、葉鞘、花及根中，而莖表現量較低。利用 OsGR2 專一性抗體進行西方墨點法結果顯示， OsGR2 顯著受到缺氮、缺鉀與缺鈣誘導表現，不同重金屬處理發現根部 OsGR2 受鎘誘導表現，在非生物逆境處理下， OsGR2 會受 ABA 、鹽、過氧化氫與缺水誘導，而植物荷爾蒙處理下激勃素、水楊酸與細胞分裂素誘導 OsGR2 表現。 PGR2/GUS 轉殖株分析顯示 OsGR2 會在葉、根、花、胚及糊粉層有較高表現。過量表現 OsGR2-GFP 融合蛋白的轉殖株水稻進一步確認 OsGR2 座落於細胞質表現。分析 RNAi 技術弱化 OsGR2 表現轉殖株顯示抑制 OsGR2 表現並減少約40%總 GR 活性，同時弱化 OsGR2 不會影響 OsGR1 及 OsGR3 之表現。功能性分析結果顯示弱化 OsGR2 會增加鹽分耐受性，反之大量表現 OsGR2 會降低耐鹽性；另外，抑制 OsGR2 表現也顯著降低植株的株高並導致稔實率下降，顯示 OsGR2 不只與鹽分耐受性有關也參與水稻莖部伸長之調控。

Glutathione reductase (GR) is an important antioxidant enzyme which responsible for reduction of oxidized glutathione (GSSG) to GSH in plant. In rice, one cytosolic and two chloroplastic GR isoforms have been identified. OsGR2 ubiquitiously expressed in all rice tissues except stem tissue, which expressed relative low levels of OsGR2 mRNA. Immunobloting analysis by OsGR2 specific antibody revealed that OsGR2 is induced by nutrient deficiency such as nitrogen, potassium, and calcium.In addition, OsGR2 is induced by abiotic stresses and plant hormone including ABA, salt-stress, H2O2, drought stress, Gibberellin, Salicylic acid and Cytokinins. Histochemical analysis of transgenic rice containing OsGR2 promoter/GUS showed that GUS accumulated in shoot, root, floret, aleurone layer and embryo. Expression of OsGR2-GFP fusion protein in rice protoplasts revealed that OsGR2 is localized at cytosol. Attenuated expression of OsGR2 by RNA interference leads to decrease 40% GR activity and did not affect the expression of OsGR1 and OsGR3. Functional analysis showed that knocked-down OsGR2 increased salt tolerance, whereas overexpression of OsGR2 reduced salt tolerance. Furthermore, knocked-down OsGR2 significantly reduced plant height and decreased the fertility of rice grains. Thus, OsGR2 is not only negative regulated with salt tolerance but also involved in promote stem elongation of rice plants.