前期研究表明黑大豆SB是一种铝敏感的大豆栽培种，SA可能通过增加SB分泌柠檬酸的能力,提高其抗铝能力。在此基础上，本项目以SB为材料，用SA或SA合成抑制剂PAC和铝共同处理SB， RT-PCR分析柠檬酸通道蛋白MATE的表达，确定SA是否调控MATE的表达诱导柠檬酸分泌;定量PCR和蛋白印迹法分析SA对铝胁迫下H+-ATPase编码基因vha2和14-3-3的表达谱和表达水平的影响；IP和Far-Western考察SA对铝胁迫下H+-ATPase磷酸化与14-3-3蛋白互作的影响，测定H+-ATPase的泵氢能力，确定SA是否通过调控H+-ATPase酶磷酸化水平及其与14-3-3蛋白互作水平，增加H+-ATPase活性，调控柠檬酸分泌。通过上述两个方面阐明SA调控Al胁迫下的柠檬酸分泌的分子机理。并在內源SA含量增加的nahG和减少的cep拟南芥突变体中，验证SA对柠檬酸分泌的调控机理

Previous study showed that black soybean SB was one of Al sensitive breeds. SA may enhance SB tolerance to Al stress by inducing citric acid secretion in SB root tip under. On the basis of the early findings,the SB was selected as the experimental materials in the programme. SB was treated with Al solution containing the SA or PAC (SA inhibitor). The RT-PCR was applied to analyse the expressive level of citric acid channel protein (MATE) to ascertain whether the citric acid increase was due to MATE expression change induced by SA.Quantitative PCR and Western-blotting was used to analyse the expression profiles and expressive level of vha2 (encoding PM H+-ATPase) and 14-3-3. The combinative ability of H+-ATPase and 14-3-3 was inspected by Co-immunoprecipitation and Far-Western, and the H+ pump activity of PM H+-ATPase were also assayed.Those date will help us confirm whether SA induced citric secretion in SB under Al stress was reslted from the enhancement of H+-ATPase phosphorylation and its interreaction with 14-3-3. On the basis of experiments mentioned above, the mechanism of SA-induced citric acid secretion in SB root tips under Al stress will be elucidated. Finally，the regulatory mechanism will be validated in nahG and cep Arabidopsis thaliana(both were mutants of SA synthesis, SA increase in nahG, SA decrease in cep).