铝胁迫下H2O2调控大豆根柠檬酸分泌的分子机理研究

The molecular mechanisms of H2O2 as signaling molecule involved in regulating the citric acid secretion of soybean root to resist Al toxicity remains unclear. The results of the preliminary study of this project suggested that Al stress increased H2O2 accumulation and reduced the plasma membrane (PM) ATPase hydrolysis activity as well as citrate secretion in Al-sensitive soybean . This study will apply H2O2 and its production inhibitors as well as its scavengers to alter H2O2 content in soybean roots under Al stress. Then further investigation will be conducted to determine whether the Al-increased H2O2 in soybean roots would play a role in regulation of the PM ATPase phosphorylation level and its interaction with 14-3-3 proteins. Using the activator and inhibitor of PM ATPase, the correlations among changes in H2O2 concentration, PM ATPase hydrolysis activity, H+ pump activity, and citrate secretion in soybean roots will also be analyzed. The function of this mechanism will be studied with H2O2 pretreated soybean roots and transgenic plant overexpressing antioxidant enzymes and a PM ATPase without phosphorylation regulation. The results of these experiments will reveal the molecular mechanism of the H2O2 regulation on PM ATPase hydrolysis activity and citrate secretion in soybean roots under Al stress. The results of this project will be valuable for further understanding the functional mechanism of H2O2 during plant response to Al stress and provide novel strategies and gene resources for genetic engineering to enhance plant Al resistance.

H2O2作为信号分子参与调控大豆根分泌柠檬酸抗铝毒分子机理仍不清楚。前期研究表明铝胁迫增加敏感型大豆根H2O2积累，降低大豆根质膜H+-ATP酶水解活性和柠檬酸分泌。本研究将使用H2O2及其抑制剂和清除剂改变铝胁迫大豆根中H2O2含量，分析铝胁迫增加大豆根中累积的H2O2是否能调控质膜H+-ATP酶磷酸化水平及其与14-3-3蛋白结合作用；用质膜H+-ATP酶激活剂和抑制剂考察大豆根中H2O2含量的变化与质膜H+-ATP酶水解活性和氢泵活性及其柠檬酸分泌的相关性; 并用H2O2预处理大豆、过表达抗氧化酶和在C末端去掉103个氨基酸的不受磷酸化调控质膜H+-ATP酶的转基因大豆研究H2O2调控作用机理。研究结果将揭示铝胁迫增加大豆根积累H2O2调控柠檬酸分泌的分子机理，丰富和加深我们对植物应答铝胁迫过程中H2O2作用机理的认识，为利用基因工程操作提高植物耐铝能力提供新的策略和基因资源。

H2O2作为信号分子参与调控大豆根分泌柠檬酸抗铝毒分子机理仍不清楚。本研究将使用H2O2及其抑制剂和清除剂改变铝胁迫大豆根中H2O2含量，分析铝胁迫增加大豆根中累积的H2O2是否能调控质膜H+-ATP酶磷酸化水平及其与14-3-3蛋白结合作用。.H2O2或铝单独处理敏感性大豆(SB)根，能使SB的RRG和H+-ATPase活性下降；AlCl3+H2O2清除剂ASA、AlCl3+DPI处理下，都能明显提高SB的RRG，有效降低SB根中H2O2含量，显著提高SB根中H+-ATPase活性。质膜ATP酶磷酸化水平及其与14-3-3蛋白互作的增强促进了质膜H+-ATPase活性的提高和柠檬酸分泌量的增加，缓解铝胁迫对SB造成的伤害。.在铝胁迫下SB经质膜H+-ATPase的激活剂FC处理后，能够减弱铝处理对其生长的抑制，增强其根尖质膜H+-ATPase磷酸化，提高其活性和柠檬酸的分泌，从而增强了SB耐铝能力；而在铝胁迫下铝耐受型黑大豆RB经质膜H+-ATPase的抑制剂AMP处理后，其根生长被显著抑制，改变铝胁迫对RB根中的磷酸化促进作用，导致其磷酸化水平、活性及柠檬酸分泌量降低，从而减弱RB的耐铝能力。强制破坏大豆根中质膜H+-ATPase的磷酸化及与14-3-3蛋白的结合，能够改变其耐铝能力。. 1μmol L-1 最佳H2O2浓度预处理SB，能显著降低植株的H2O2和MDA含量，诱导SB的SOD和POD基因表达导致提高SB抗氧化酶POD和SOD酶活性，明显增加质膜H+-ATPase和14-3-3蛋白的表达量，增强质膜ATP酶与14-3-3蛋白的互作，提高H+-ATPase活性和柠檬酸分泌量，从而提高SB铝抗性。 .转抗氧化酶GmCAT基因能提高转基因烟草CAT酶活性，降低烟草H2O2含量达75%，过低的H2O2含量影响其作为信号分子参与调控植物生长发育的功能，导致烟草生长缓慢。转GmSOD基因能提高烟草SOD酶活性，维持根中低 H2O2含量。低浓度H2O2作为信号分子促进质膜H+-ATP酶的磷酸化水平及其与14-3-3蛋白的结合，提高烟草根质膜 H+-ATPase水解活性和 H+泵活性，促进柠檬酸的分泌，提高抗铝毒能力。在烟草中过表达持续激活型的外源ΔGHA2基因，不能提高转基因烟草磷酸化水平，但能显著提高其质膜H+-ATPase的活性和柠檬酸的分泌，提高耐铝毒能力