摘要
以NN烟草(NicotianatabacumL.)品种“SamsunNN”(与烟草花叶病毒(TMV)发生非亲和互作)和普通烟草(N.tabacumL.)“3002”品种(与TMV发生亲和互作)叶片为材料,采用差速离心法和两相法制备密闭的正向型质膜(PM)囊泡。用SOD敏感的依赖于NADPH的Cytc的还原表示质膜NADPH氧化酶产生超氧阴离子(O-·2)的活性。加入0.01%TritonX_100可使酶活性提高约80%,证明NADPH结合位点在质膜电子载体蛋白胞质结构域上,而O-·2在质膜外侧生成,在反应过程中发生了跨质膜的电子传递。质膜NADPH氧化酶的活性可部分地被哺乳动物NADPH氧化酶的专一性抑制剂DPI(diphenyleneiodonium)抑制,最大抑制达70%。受TMV侵染后,NN烟草NADPH氧化酶活性显著升高,而普通烟草的酶活性基本不变。
The plasma membrane NADPH oxidase and its regulatory role in the production of reactive oxygen species (ROS) in tobacco ( Nicotiana tabacum L.)_tobacco mosaic virus (TMV) interaction was examined by using tobacco cv. “Samsun NN' (incompatible with TMV, containing the N gene for resistance to TMV) and tobacco cv. “3002' (compatible with TMV) as experimental materials. Plasma membrane (PM) vesicles were isolated from leaves of tobacco by a biphasic aqueous system. The membrane preparations were sealed, highly purified and largely in right_side_out orientation as detected by marker enzyme assays and latency studies of the PM marker, vanadate_sensitive ATPase with non_ionic detergent Triton X_100. The oxidase activity was assayed by the rate of SOD_sensitive Cyt c reduction in PM system. The oxidase activity could be increased about 80% when adding 0.01% Triton X_100 in the reactive system. This result showed that the binding_site of NADPH was on the cytosolic side of the plasma membrane and the production of O -· 2 is on the apoplastic side. DPI (diphenylene iodonium), a specific inhibitor of the NADPH oxidase in neutrophils, also inhibited the NADPH oxidase activity in tobacco. Furthermore, the oxidase activity increased in incompatible interaction, but not in compatible interaction. The role of NADPH oxidase in the production of reactive oxygen species and stimulation of hypersensitive reaction were discussed.
基金
国家自然科学基金
植物病虫害生物学国家重点实验室基金资助项目
关键词
NADPH
氧化酶
烟草
烟草花叶病毒
质膜
NADPH oxidase, Superoxide radical anion, Tobacco, Tobacco mosaic virus, Plasma membrane
