磷、铝和低pH值对大豆苹果酸转运子GmALMT1的分子调控机理

Low phosphorus (P) availability, aluminum (Al) toxicity and low pH often coexist in acid soils, and therefore are considered as the three major stresses limiting crop growth on acid soils. To date, few studies have addressed the coordinated effects of low pH, Al toxicity and low P availability on crop growth. Our previous studies showed that root malate exudation regulated by GmALMT1 was one of the major mechanisms for soybean adaptation to acid soils. In the present study, we plan to clarify the regulation mechanisms of GmALMT1 by the coordination of P, Al and low pH at both transcriptional and post-translational levels. The cis-elements in the GmALMT1 promoter will be analyzed using the transgenic soybean hairy-roots carrying promoter-GUS fusions. The up-stream transcription factors will be identified by yeast one-hybrid system. EMSA will be utilized to further verify the interactions between the GmALMT1 promoter and the transcription factors. Moreover, single point mutations in amino acid residues predicted to be phosphorylation sites of GmALMT1 will be generated. The transport properties of these mutations will be examined physiologically and electrophysiologically by using transgenic tobacco suspension cells and two-electrode voltage-clamp technique, respectively. Our results might establish the molecular regulation network of the coordinative responses of GmALMT1 to P, Al and low pH, so as to provide the valuable theoretic basis for crop improvement in acid soils through biotechnological approaches.

低磷、铝毒和低pH值是酸性土壤中限制作物生长的主要因素。我们前期工作表明，根系分泌苹果酸是大豆适应酸性土壤的重要生理机制，且此苹果酸的分泌主要受苹果酸转运子GmALMT1的调控。本项目拟在转录和蛋白质水平上，解析磷、铝和低pH值调控GmALMT1的分子机制。首先，通过大豆转化体系，利用GmALMT1启动子融合GUS报告基因，分析与磷、铝和低pH值响应有关的顺式作用元件；再利用酵母单杂系统，克隆调控GmALMT1表达的潜在转录因子基因；通过酵母单杂和EMSA技术，检验所克隆的转录因子与GmALMT1启动子的互作情况。通过点突变技术将GmALMT1蛋白的潜在磷酸化位点突变，利用转化烟草悬浮细胞和爪蟾卵母细胞双电极电压钳技术，分析蛋白磷酸化修饰对GmALMT1蛋白转运活性的影响。研究结果将初步构建调控GmALMT1协同响应磷、铝和低pH值的分子网络，为培育适应酸性土壤的作物品种提供理论依据。

低磷和铝毒是酸性土壤中限制作物生长的主要因素。苹果酸转运子GmALMT1调控的根系苹果酸分泌是大豆适应磷铝胁迫的重要机制。为了解析磷铝协同调控GmALMT苹果酸转运子的分子机理，本研究克隆了其上游转录因子，初步明确了各转录因子对GmALMT1表达的调控作用。另外，对大豆其他GmALMT基因家族成员响应低磷胁迫的表达模式也进行了分析，初步阐明了GmALMT苹果酸转运子在大豆活化和利用难溶磷中的重要作用。研究结果为培育适应酸性土壤的大豆品种提供了理论依据和重要的基因资源。