菌根特异铵转运蛋白介导的植物-AM真菌共生界面上的氮素转运机制研究

Arbuscular mycorrhizal (AM) symbiosis brings together the roots of plant and the fungi of the phylum Glomeromycota, and improves their mutual benefits for exchanges of mineral nutrients and photosynthetically fixed carbon. Besides phosphorus (P) nutrient, AM can play an important role for the acquisition of nitrogen (N) by plant roots. However, the solid evidences at the physiological and molecular levels are still missing to support the contribution of mycorrhizal pathway to plant N uptake (Myc-N pathway). In the previous work, we established an advanced system to evaluate the Myc-N pathway in soil-cultured maize plants based on 15N-labelling N strategy in AM-compartments, and showed that a significantly higher 15N abundances in shoots of maize plants grown with 15N- mycorrhizal compartment. This suggested an important contribution of mycorrhizal to N transport into maize plants. Meanwhile, we isolated the myc-specific each of AMT and NIP gene, which might be involved in N transport from mycorrhizal to plant root cells in a form of ammonium. Here, we aim to characterize these two candidate genes by investigating 1) the cellular and sub-cellular location in maize roots when colonized by AM; 2) the transport properties in terms of ammonium substrate when heterogously expressed in oocytes, and yeast or Arabidospsis in which the multiple AMTs were disrupted; 3) the function for N-transport from AM to plant cells in vivo using the transgenic maize RNAi lines in which the target genes expression were silenced. There results will not only provide the physiological evidence to support the contribution of N transport during mycorrhizal symbiosis, and also reveal the essential roles of plasma-membrane localized membrane transporters in arbusculated cortical cells in NH3/NH4+ transport to plant roots.

丛枝菌根真菌（AM）与植物根系形成互惠互利的菌根共生体，实现植物和真菌之间的资源交换。有证据表明AM真菌对植物氮素吸收有重要作用，但其生理贡献大小及共生界面上的氮素转运机制尚不清楚。我们前期研究建立了土培条件下菌根室15N标记的实验体系，证实了玉米菌根氮素吸收途径的显著贡献；并筛选分离到菌根特异诱导的玉米铵转运蛋白（AMT）和水通道蛋白（NIP）基因，可能介导了铵从菌根向根系的传递。本项目拟深入研究两个候选基因在根中的组织和亚细胞定位；在卵母细胞、酵母和拟南芥AMT多缺失突变体中异源表达来研究铵转运特性；在玉米RNAi转基因系中沉默基因的表达，利用建立的玉米毛根-菌根培养体系和土培菌根室体系，研究它们在介导菌根氮素吸收过程中的作用。研究结果将最终明确氮素在根系-菌根共生界面上以铵的方式运输，及其菌根特异诱导的AMT和NIP 蛋白的功能机理，从而在分子上阐明植物根系菌根氮素吸收途径的机理

丛枝菌根真菌（AM）能够与植物根系形成互惠互利的菌根共生体，从而保障植物和真菌之间所需资源（碳、磷、和氮等）的交换。丛多证据表明，除了根系的直接吸收途径之外，AM菌根介导的氮素吸收途径对植物氮营养效率也有重要贡献。根系-菌根共生界面上氮素转运过程被认为是菌根氮素吸收途径的关键，但其分子机制还尚不清楚。本项目以玉米为研究对象，筛选分离得到一个菌根特异诱导表达的玉米铵转运蛋白基因ZmAMT3;1。原位杂交和启动子驱动的报告基因分析发现，该基因转录本和启动子活性主要定位在AM菌根侵染的玉米皮层细胞中。玉米原生质体中瞬时表达ZmAMT3;1-GFP融合基因证明ZmAMT3;1是质膜蛋白，而且进一步分析ZmAMT3;1pro:gene:GFP转基因玉米发现ZmAMT3;1蛋白特异性定位在植物-AM菌根共生界面的周膜上。酵母功能互补与15N标记铵吸收试验发现，ZmAMT3;1蛋白能够介导高亲和力的铵转运过程，而且转运活性还受到蛋白C末端磷酸化修饰的调控。在卵母细胞中异源表达ZmAMT3;1蛋白没有检测到相应的电流，但可以检测到15N标记铵的累积，说明是中性氨分子（NH3），而不是铵离子（NH4+）被转运。利用创建的ZmAMT3;1-RNAi转基因玉米沉默系，通过玉米根室-菌丝室分离的土培体系，并结合菌丝室15N铵态氮标记技术，发现与野生型对照相比，ZmAMT3;1-RNAi转基因系完全丧失了依赖菌根途径的氮吸收能力。并且，在菌丝室氮素为主要氮素来源的培养条件下，ZmAMT3;1-RNAi转基因系的吸氮量要显著低于对应的野生型，但吸磷量仍保持不变。因此，以上的遗传、生化和分子证据充分表明，定位于根系-菌根共生界面周膜上的高亲和铵转运蛋白ZmAMT3;1介导了氨从菌根向根系的传递过程。这些研究结果不仅阐明了植物依赖菌根介导的氮素吸收途径的分子机理，也为农业生产中有效利用植物-菌根互作提高作物氮效率提供了理论支撑。