铝诱导荞麦根系有机酸分泌的分子机制研究

Aluminum (Al) toxicity represents one of the major limiting constraints to plant growth and development on acidic soils. Al-induced organic acids secretion (mainly citric acid, malic acid, and oxalic acid) is a widely adopted and very important mechanism by which plants deal with Al toxicity. To date, the genes responsible for Al-induced citric acid secretion and malic acid secretion have been isolated in a number of plant species. However, the gene related to Al-induced oxalic acid secretion has not been identified in any species. In addition, it seems that the entrance of Al into root cell symplast is unavoidable even to those plant species that resist Al toxicity mainly by means of exclusion mechanisms. Thus, it is worth to note regarding how to improve the translocation efficiency of Al from root to shoot, and as a consequence, detoxify Al within leaf vacuoles. It has been demonstrated that roots of buckwheat secret oxalic acid into rhizosphere and citrate into xylem when suffering from Al stress. Based on these findings, in this project, we aim to identify the key genes involved in Al-induced oxalic acid secretion and citric acid secretion through comparative analysis of transcriptomic and proteomic responses of buckwheat root apex to Al stress. By means of promoter cloning and analysis, as well as yeast-one-hybrid screening, we try to clarify the transcriptional regulation of these genes. Transgenic approaches will help us to explore whether high and efficient Al resistance plants could be constructed through modification of upstream signal transduction pathways. For the first time, this project will investigate the molecular mechanism of Al-induced organic acids secretion from buckwheat roots, and will further our understanding of Al tolerance mechanism in higher plants. Furthermore, the achievements of this project will also provide scientific guidance for the fields concerning signal transduction of Al stress, evolution of Al tolerance genes, and improvement of Al tolerance through biotechnology.

铝毒是限制酸性土壤作物生产的主要因子之一。铝诱导植物根系分泌有机酸（柠檬酸、苹果酸和草酸）是较普遍和重要的耐铝机制。目前关于铝诱导柠檬酸和苹果酸分泌到根际的基因已在一些物种中被克隆，而诱导草酸分泌的基因还未在任何物种中被报道。此外，由于铝进入根细胞内的不可避免性，将铝从木质部转运到地上部而在叶片解铝毒的重要性也值得关注。本项目以铝诱导荞麦根系分泌草酸到根际和分泌柠檬酸到木质部为前提，结合转录组和蛋白质组分析，挖掘调控铝诱导荞麦根尖有机酸分泌的关键基因；通过启动子分析和酵母单杂交筛选，明确关键基因的转录表达调控机制；通过转基因功能验证，探索通过改造上游信号途径使植物更好适应酸性土壤的可能性。本项目首次系统的开展铝诱导荞麦有机酸分泌的分子机制，可望对植物耐铝机制作出更新和更全面的阐述，为研究铝胁迫信号转导途径、耐铝基因遗传转化、以及通过生物工程手段提高作物耐铝性提供了科学依据。

铝是地壳中最丰富的金属元素。在酸性土壤环境中，离子态铝溶解进入土壤溶液，对大多数植物的生长发育造成严重影响。因此，研究植物抗铝机制，遗传改良植物抗铝性是保障酸性土壤地区粮食安全的有效途径之一。荞麦是一种适应酸性铝毒土壤的作物，它在酸性铝毒土壤上生长良好，表明进化出了特殊的抗铝机制。前期的研究基础表明，荞麦根系分泌有机酸是其抗铝的重要机制。但是调控荞麦根系有机酸分泌的分子机制仍不是很清楚。本研究项目围绕荞麦对铝胁迫的响应，从转录组分析、基因挖掘以及功能分析等方面开展荞麦抗铝的分子机制。主要研究结果如下：利用转录组测序，解析了荞麦根尖和叶片响应铝胁迫的分子机制，确定了关键转运蛋白基因，为后续功能分析奠定了基础，相关成果发表于Frontiers in Plant Science（2017）和International Journal of Molecular Sciences（2017）上。基于转录组分析结果，克隆并鉴定了两个关键转运蛋白基因FeSTAR1和FeALS3，明确了它们通过调控细胞壁代谢参与荞麦抗铝，相关成果发表于Plant Soil（2018；2019）上。为了深入分析草酸代谢机制，克隆了荞麦参与草酸乙酰化降解途径的关键酶基因FeAAE，并对其铝胁迫的转录表达进行了研究，成果发表于Plant Signaling & Behavior上（2017）。为了进一步挖掘参与草酸分泌过程中的关键因子，分析了番茄根尖响应铝胁迫的转录组，挖掘了相关转运蛋白基因，并对番茄AAE家族进行了系统分析。通过蛋白原核表达和生化分析，明确了番茄AAE蛋白对草酸降解的底物专一性，进一步利用Crispr/cas9基因编辑技术，获得了相关基因编辑材料。