铝胁迫增强耐铝蚕豆根质膜H+-ATPase活性及柠檬酸分泌的分子机理研究

Organic acid exudation is an important mechanism of plant Al resistance. Numourous studies have shown that elevation of plasma membrane (PM) H+-ATPase activity can enhance citrate secretion in plants under low P and Al stress. Enhancement in PM H+-ATPase activity via the interaction between 14-3-3 protein and PM H+-ATPase is involved in reponses to many abiotic stresses. The preliminary studies of this project showed that citrate secretion and PM H+-ATPase activity was enhanced in an Al-resistant Yunnan broad bean (Vicia faba L.) cultivar (8363) under Al stress. The transcriptions of PM H+-ATPase encoding gene (vha2) and a 14-3-3 protein encoding cDNA (vf14-3-3b) were also up-regulated by Al. Thus, it is speculated that Al stress might increase PM H+-ATPase activity and citrate secretion via an enhancement in the expression and interaction of PM H+-ATPase and 14-3-3 protein. Using YD as the material, real time RT-PCR and Western analysis will be performed to investigate transcription profiles and expressions of vha2 and vf14-3-3b under Al stress. The effects of Al stress on the interaction between PM H+-ATPase and 14-3-3 protein will be investigated by immunoprecipitation and Far-Western analysis. The relationship between PM H+-ATPase activity and citrate secretion will be examed by changing the interaction between PM H+-ATPase and 14-3-3 protein via inhibitor and activator. The results obtained from these experiments will clarify the molecular mechanism for the interaction between PM H+-ATPase and 14-3-3 protein to regulate citrate secretion in response to Al stress in YD. The function of this mechanism will also be verified in transgenic tobacco. The resuslts of this project will be helpful for us to further understand the mechanism of plants in response to Al stress and provide novel strategy and gene resources for genetic engineering to enhance plant Al resistance.

本项目前期研究结界表明铝胁迫可能通过增强质膜(PM) H+-ATPase和14-3-3b蛋白的表达及结合能力来提高云南耐铝蚕豆（YD）PM H+-ATPase的活性及蚕豆分泌柠檬酸的能力。本研究以YD为材料，用定量PCR和Western分析考察铝胁迫下PM H+-ATPase和14-3-3b的表达谱和表达水平，免疫共沉淀和Far-Western分析考察铝对PM H+-ATPase与14-3-3蛋白互作的影响，通过抑制剂和激活剂改变铝胁迫下PM H+-ATPase和14-3-3蛋白的结合能力，考察PM H+-ATPase活性和YD柠檬酸分泌的相关性，阐明14-3-3蛋白和PM H+-ATPase的互作调控蚕豆柠檬酸分泌应答铝胁迫的分子机理，最后在转基因烟草中验证这个机理是否发挥作用。研究结果有助于加深对植物应答铝胁迫机理的了解，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。

本研究以铝耐受型蚕豆YD和铝敏感型蚕豆AD为研究材料，研究结果说明铝胁迫下蚕豆根尖质膜H+-ATPase活性和柠檬酸分泌均呈现显著性正相关；铝胁迫对YD根中质膜H+-ATPase(vha2)和14-3-3b表达的诱导作用比AD强烈；铝胁迫增加YD根尖VHA2的磷酸化水平及其与14-3-3b蛋白的结合能力，而在AD根中却产生抑制作用。在铝胁迫下镁的存在增加铝诱导质膜H+-ATPase的活性和柠檬酸的分泌，缓解铝对YD根尖生长的抑制作用。镁离子对铝胁迫下YD根中vha2的表达没有诱导作用，这说明铝胁迫处理YD根中产生一个上调vha2表达的信号转导途径，而镁处理YD根中并没有产生相似的信号转导途径。在体外试验中证实镁可以增加质膜H+-ATPase与14-3-3b的结合。体内试验结果证实镁离子在蛋白水平上通过增加14-3-3b与VHA2的相互作用来增强质膜H+-ATPase的活性。构建H+-ATPase与14-3-3的过量表达（ΔGHA2，SGF14a）和RNAi干扰（pma4、tb14-3-3b）载体，转化烟草产生H+-ATPase与14-3-3的过量表达和抑制表达转基因株系。研究结果说明在烟草中SGF14a的过量表达能够增加烟草对铝胁迫的耐受性，抑制tb14-3-3b的表达明显降低转基因烟草对铝胁迫的耐受性。在烟草中过量表达SGF14a产生大量的14-3-3蛋白与磷酸化质膜H+-ATPase结合，不仅能够增强质膜H+-ATPase的活性和H+的分泌作用，并且还能够拮抗铝对其活性的抑制，增加烟草根尖柠檬酸的分泌量。而在烟草中抑制tb14-3-3b的表达时产生相反的结果。在烟草中过量表达ΔGHA2显著提高转基因烟草根中质膜H+-ATPase的活性和根尖柠檬酸的分泌量及耐铝能力；而抑制pma4的表达显著减少转基因烟草根尖磷酸化质膜 H+-ATPase与14-3-3蛋白的结合，同时也显著降低烟草根尖质膜H+-ATPase的活性和H+分泌能力及柠檬酸的分泌作用，增加铝胁迫下烟草根内H2O2的积累，降低烟草对铝胁迫的耐受性。这些研究结果不仅丰富和加深我们对植物应答铝毒胁迫机理的了解和认识，还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。