蒺藜苜蓿液泡磷转运体的功能鉴定与调控机理研究

液泡磷对于维持细胞的磷素平衡至关重要，作为重要的豆科牧草，苜蓿的液泡磷转运体及其调控途径完全不清楚。研究表明，SPX-MFS家族蛋白是植物的液泡磷转运体，但它们的转运方向及活性各有不同。我们将以蒺藜苜蓿为材料，采用拟南芥液泡磷转运体vpt1突变体和VPT1过表达植株中异源表达来筛选苜蓿MtSPX-MFS家族中的功能蛋白，再用苜蓿突变体对MtSPX-MFS的分子功能进行深入鉴定。基于MtSPX和MtSPX-RING家族蛋白对MtSPX-MFS的活性和蛋白水平调控的推测。拟通过酵母双杂和双分子荧光互补（BiFC）试验证实蛋白-蛋白互作关系。利用DEX(地塞米松)诱导、RNAi等方法控制目的基因的表达，结合Western-blot、31P-NMR、膜片钳等技术为上述推测提供直接证据。本研究不仅可从液泡水平上揭示苜蓿的磷素平衡机制，也可为苜蓿的磷素高效利用及分子育种提供科学依据。

Medicago is one of the most important leguminous forages, and also phosphate preference plant. The vacuolar phosphate pool is essential for maintaining phosphate homeostasis in plant cells, whereas the vacuolar phosphate transporters and their regulation pathways are completely unclear in Medicago plants. According to previous studies, the SPX-MFS family members are likely to be plant vacuolar phosphate transporters, but their transport directions and activities are diffident. We plan to identify the molecular function of MtSPX-MFS in Medicago truncatula by heterologous expression in Arabidopsis vpt1 (vacuolar phosphate transporter 1) mutant and VPT1 over-expression (VPT1-OEX) plants, and by medicago mutant. On the other side, we speculate that members in MtSPX and MtSPX-RING families have regulations on protein activities and levels of MtSPX-MFS respectively. We plan to identify the protein-protein interaction by yeast two-hybrid and bimolecular fluorescence complementation (BiFc) methods, and identify the molecular function of protein interaction by Dexamethasone (DEX) inducible expression system construction, RNAi, Western-blot, 31P-NMR, Patch-clamp and other methods. This study not only could uncover the phosphate homeostasis mechanism of Medicago plants on vacuole aspect, but also could provide more paths for engineering better phosphate efficient Medicago plants.