The Physiological And Molecular Mechanisms Of The Effect Of Hydrogen Peroxide Produced By The Sensitive Black Soybean Under Aluminum Stress

Acidic soil accounts for30%of arable soil in the world, mainly distributed in developing countries including South Africa, Central Asia, Southeast Asia and so on, which have great food demand(Sasaki et al.2004). In China, acidic soil are mainly distributed in tropical, subtropical regions and Yun Guichuan, which are in the south of the Yangtze River covering an area of up to2040000hectares. The aluminum toxicity in acid soil is the major factor limiting the growth and yield of crops. In this study, aluminum sensitive black soybean (SB) were taken as material, investigating the physiological and molecular mechanisms of the effect of hydrogen peroxide produced by the sensitive black soybean under aluminum stress. The main results are as follows:1. The total RNA was extracted from the root of SB, PCR target gene fragment of14-3-3was acquired by reverse transcription, then by the TA cloning, enzyme digestion, and sequencing, joined to the prokaryotic expression vector pGEX-4T-1, to construct the recombinant plasmid pGEX-4T-1-14-3-3was transformed into bacteria, were induced and expressed in Escherichia coli BL21. Under the1mM IPTG, different temperature and time gradient to find out the optimum induced condition, finally determined the best conditions for inducing the expression of recombinant protein of pGEX-4T-1-14-3-3was induced by1mM IPTG,28℃, for8h, and the detection of SDS-PAGE protein expression mainly in the soluble expression, purification of recombinant protein was acquired by GST affinity chromatography, which concentration of the pure objective recombinant protein was1mg/mL, the total content was4mg, and for the antibody preparation.2. By using H2O2or aluminum treatment alone, with the H2O2scavenger ASA, the inhibitor of ABA synthesis, sodium tungstate (sodium tungstate), NADPH oxidase inhibitors DPI and aluminum stress at the same time processing and secretion of SB root, determination of root RRG and H2O2content and citric acid level, the relationship between sensitivity and H2O2content of SB on aluminum, if the increase of H2O2content the need to increase the synthesis and the activity of NADPH oxidase ABA, aluminum stress correlation SB root in the production and accumulation of H2O2and citric acid secretion. At the same time, SB analysis of root plasma membrane ATP ATPase and14-3-3protein expression level, plasma membrane ATP enzyme phosphorylation and14-3-3protein interaction activity and pump hydrogen ability level, plasma membrane ATP enzyme, correlation study under aluminum stress SB root H2O2content and plasma membrane H+-ATP enzyme phosphorylation and14-3-3protein interaction level, plasma membrane H+-ATP the enzyme activity.3. Through the use of plasma membrane H+-ATP enzyme activator FC and MgC12with aluminum at the same time processing SB root, the determination of citric acid secretion, RRG, H2O2content, and the analysis of the SB root plasma membrane ATP ATPase and14-3-3protein expression level, plasma membrane ATP enzyme phosphorylation and14-3-3protein interaction activity and hydrogen capacity level, the accumulation effects of H2O2in SB root to the plasma membrane ATP activity and citrate exudation under aluminum stress.4. Through the experiment to determine the optimal concentration of H2O2pretreatment is1μmol·L-1, and then use the optimal concentration of H2O2pretreatment with SB induced the expression of antioxidant enzymes, and then use the aluminum stress to pretreat SB, determined the SB root RRQ the H2O2content, and the analysis of the SB root plasma membrane ATP ATPase and14-3-3protein expression level, plasma membrane ATP enzyme phosphorylation and14-3-3protein interaction activity and pump hydrogen ability level, plasma membrane ATP enzyme, further verify the molecular mechanism of plasma membrane ATP activity and citrate exudation resulted from the accumulated effects of H2O2in roots of SB under aluminum stress.