采用溶液培养的方法,以Al敏感型黑大豆(SB)为材料,研究外源添加水杨酸(SA)对Al胁迫下SB根生理生化指标和Al胁迫相关基因表达的影响,探讨外源SA缓解黑大豆Al毒害的分子机理。结果表明:低浓度(10,20!mol·L-1)SA缓解了Al毒引起的根伸长抑制,其中20!mol·L-1SA的缓解效果更明显;而高浓度SA和SA合成抑制剂多效唑(PAC)处理加重了Al毒引起的根伸长抑制;外源添加20!mol·L-1SA使Al胁迫下SB根尖MDA和H2O2含量下降,根系分泌物中柠檬酸的含量约是单独Al处理的2倍;基因表达分析表明外源添加20!mol·L-1SA促进Al胁迫下SB根中12个14-3-3亚型、质膜H+-ATP酶和柠檬酸通道蛋白(MATE)基因的表达;免疫共沉淀的结果表明外源添加20!mol·L-1SA提高了Al胁迫下质膜H+-ATP酶蛋白磷酸化水平及其与14-3-3蛋白结合能力;外源添加PAC的效果与外源添加SA的相反。外源SA可能通过诱导Al胁迫下MATE表达,提高14-3-3蛋白和质膜H+-ATP酶基因蛋白水平和互作,增加柠檬酸的分泌,增强黑大豆对Al毒害的耐受性。
A method of solution culture to Al-sensitive black soybean(SB) was used to study the effects of exogenous salicylic acid(SA) on the physiological parameters and Al stress-related gene expression in SB root under Al stress.The aim of the study is to explore the molecular mechanisms of exogenous SA alleviating Al toxicity in SB.The results showed that a low concentration(10 and 20 !mol·L-1) of SA alleviated inhibition of root elongation caused by Al toxicity,wherein the alleviation effect of20 !mol·L-1SA more significant.However,high concentration of SA and SA synthesis inhibitors paclobutrazol(PAC) increased inhibition of root elongation caused by Al toxicity.Exogenous 20 !mol·L-1decreased the MDA and H2O2 content in SB under Al stress,and the citric acid content in root exudates was about twice as higher as that of single Al treatment.Expression analysis showed that exogenous 20 !mol·L-1SA enhanced the expression of the 12 substype of 14-3-3 isoforms,plasma membrane H+-ATPase and multidrug and toxic compound extrusion transporter(MATE) gene in SB under Al stress.The results of the co-immunoprecipitation showed that exogenous 20 !mol·L-1SA improved the levels of protein phosphorylation for plasma membrane H+-ATPase and its binding capacity with 14-3-3 protein under Al stress.The effect of exogenous PAC treatment was opposite with SA.It was suggested that exogenous SA may enhance the SB tolerance to Al stress by inducing MATE expression and enhancing 14-3-3 and plasma membrane H+-ATPase protein levels and interaction between them to increase citric acid exudation.