水杨酸增加铝敏感型黑大豆SB根系柠檬酸分泌的机理研究

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).

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

有机酸分泌是缓解Al毒害的重要生理机制。黑大豆SB是一种铝敏感大豆，发现SA+Al处理SB，能缓解Al诱导的根伸长抑制，诱导根系柠檬酸的分泌。RT-PCR表明SA诱导Al胁迫下SB根系多种14-3-3蛋白，H+-ATP酶和MATE的转录。Western Blotting发现SA+Al处理下14-3-3f及14-3-3j蛋白的表达显著高于Al处理，SA诱导了Al胁迫下上述二个蛋白的表达。免疫共沉淀表明SA+Al处理下14-3-3f与磷酸化H+-ATP酶的结合能力显著高于Al处理，而14-3-3j与磷酸化H+-ATPase的结合能力与Al处理无显著差异。酶活测定表明，SA+Al处理下SB根中H+-ATP酶和H+泵活性显著高于Al处理和对照。SA+Al处理中，添加抑制剂抑制14-3-3f与H+-ATPase的结合，H+-ATPase和H+泵活性相应降低。综上所述，SA通过诱导14-3-3f的表达，增强14-3-3f与磷酸化H+-ATP酶的互作水平，增加H+-ATP酶和H+泵活性，诱导SB柠檬酸分泌。300µ M Al 胁迫下，拟南芥突变体npr1-1有机酸分泌量大于nahG， 内源SA含量与有机酸分泌正相关。免疫共沉淀表明npr1-1中 PAC+Al处理下14-3-3与磷酸化H+-ATP酶互作水平显著低于Al处理，nahG中SA+Al处理下上述两个蛋白的互作水平高于Al处理。酶活分析表明，SA也能够增强Al胁迫下拟南芥H+-ATPase和H+泵活性。SA诱导拟南芥有机酸的分泌可能与其增强14-3-3与磷酸化H+-ATPase蛋白互作水平，增加H+-ATPase和H+泵活性有关。RT-PCR和Western Blotting分析表明，抑制剂PAC减少了Al胁迫下npr1-1中 14-3-3φ及14-3-3o基因和蛋白的表达。免疫共沉淀表明两种突变体中Al胁迫下14-3-3φ及14-3-3o与磷酸化H+-ATP酶的互作水平与对照无差异。添加SA或PAC后，二者的互作能力与Al和对照也无显著差异。在拟南芥中，SA并不是通过调控14-3-3φ及14-3-3o与磷酸化H+-ATPase的互作水平，诱导有机酸分泌。拟南芥中SA通过调控何种亚型14-3-3蛋白，诱导有机酸分泌仍需研究。本研究为SA缓解植物Al毒害，调控有机酸分泌提供了新的研究模式