| Oxidative stress is the secondary stress in plants responsed to stresses and NADPH oxidase is mostly accepted as responsible for the main course of ROS productions. In our experiments, the fully cDNA of tobacco NADPH oxidase was cloned according to the published sequenced, and protein sequence was analyzed by bio-information methods. The main function domain of plant NADPH oxdiase was observed on our protein sequence, and protein phosphatization maybe involved in the regulation of NADPH oxidase activity because many potential phosphatization sites were found by Protein modification analyse.The expression protein from a 500bp segment of NADPH oxidase C terminus expressed in E.coli by constructing the PET-NAO expression plasmid was purified through the Qiagen Ni-NTA protein purification systems and used to gain polyclone antibody from rabbit. The value of polyclone antibody reached 1:1000. Meanwhile, we constructed the overexpression (NAO) and two RNAi (NAOa, NAOb) vectors of NADPH oxidase promoted by CaMV 35S and transform them into tobacco. The transgenic tobaccos were identified via genome DNA PCR, RT-PCR and Western blot. Ten NAO lines had the higher level of transcription and translation of NADPH oxidase, and the transcription and translation of NADPH oxidase was inhibited in eight NAOa lines and twelve NAOb lines.The transcriptions of antioxidative enzymes were induced in Arabidopsis by foreign H2O2. But it is unclear that effect of endogenic H2O2 on the antioxidative enzymes. The activity of CAT, POX, APX, GR and SOD was detected in NADPH oxidase transgenic lines. Data showed us that there was higher level of antioxidative enzymes in overexpression transgenic tobacco in comparison with the control. On the contrary, the activity of antioxidative enzymes was partly inhibited. However, there were no markedly varieties on the aspect of growth and development between transgenic and control lines. We predicted that there was a new balance between the productions and eliminations of ROS in transgenic tobacco.The ROS productions were inhibited by DPI, a specific inhibitor of NADPH oxidase, in Arabidopsis under salt stress. But it is unclear that the effect of salt stress on the activity of NADPH oxidase. The activity of plasmamembrane NADPH oxidase from tobacco under NaCl stress was detected by the method of Cytochrome C reduced. Data showed us that NADPH oxidase was markedly actived as the tobacco was treated with lower concentrations (100-200mM) of NaCl or shorter time (1-6h). NADPH oxidase involved in the salt-induced ROS productions. However, Treatment with higher concentrations NaCl(>200mM) or longer time(>6h) decreased the activity of NADPH oxidase.The growth and development of transgenic was observed under long-term NaCl stress, overexpression transgenic tobacco emerged the inhibitor of growth and development treated with 200mM NaCl two months in comparison with the control. The activity inhibitor of antioxidativeenzymes and higher lipid peroxidation level were observed in overexpression transgenic tobacco responsed to long-term salt stress. On the contrary, the RNAi transgenic tobacco grew better than the control and overexpression transgenic tobacco. Lower activity inhibitor of antioxidative enzymes and lipid peroxidation level were detected in RNAi transgenic tobacco. We also detected the seed germination of overexpression plants on MS solid medium contained 100mM NaCl, the inhibitory effect of NaCl on the seed germination of overexpressions plants was more obviously than wild type.Excess Cu2+ induced the elevation of NADPH oxidase acitvity in wheat roots. However, it is unclear that the regulation mechanisms of Cu2+-induced elevation of NADPH oxidase activity in tobacco suspension cells. Our data showed that ROS produced from NADPH oxidase, regulated by Cu2+-induced PLD, was the main source of the ROS productions in BY-2 suspension cells under Cu2+ stress. We predicated that like mammalian NADPH oxidase, plant NADPH oxidase was also regulated by PLD and its production PA.
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