OsAAP8调控水稻氮素营养利用的生理和分子机制

Amino acids are the major transport form of nitrogen in rice. Leaf nitrogen remobilization via phloem is crucial for growth of young tissues and seed development. However, the details of amino acids remobilization from rice source leaves and the key transporters responsible for this process are yet unclear. In this study, we selected OsAAP8 as the candidate transporter according to multiple characteristics of amino acids remobilization from leaves. The expression level of OsAAP8 was increased coincidentally with the strength of amino acids remobilization from source leaves. Green fluorescent protein analysis using transient expression and transport activity identification in yeast showed that OsAAP8 localizes to plasma membrane and is a functional amino acids uptake transporter. Microarray data revealed that OsAAP8 is probably expressed in vascular tissues. Those evidences indicate that OsAAP8 plays a key role in amino acids remobilization from leaves. Interestingly, a relative highly expression level of OsAAP8 was also detected in roots and nodes by qRT-PCR analysis, which indicates that OsAAP8 is also involved in many other processes of distribution of amino acids within rice plants. We also constructed OsAAP8 related knockout, over-expression and promoter-β-glucuronidase fusions transgenic rice plants (Nipponbare), and then we will combine physiological, biochemical and molecular biology techniques to uncover the mechanisms of OsAAP8 mediated distribution of amino acids within rice plants, and further to elucidate the mechanisms of nitrogen utilization regulated by OsAAP8. In addition, the results from this project will also provide theoretical basis and genetic resources for increasing nitrogen use efficiency in rice.

氨基酸是水稻体内转运利用的主要氮素形态，叶片氮素以氨基酸形式经韧皮部的再利用是幼嫩组织和种子发育所需氮素的重要来源。然而，水稻叶片氨基酸再转运的详细过程和负责的转运蛋白都还不清楚。我们依据叶片氨基酸再利用的特征对水稻氨基酸转运蛋白家族基因进行筛选，筛选到了OsAAP8。OsAAP8在叶片氨基酸再利用的关键期被诱导高表达；编码质膜定位蛋白；能将氨基酸转运至胞内；可能表达在维管组织。这些都指向OsAAP8参与了叶片氨基酸的再转运。有趣的是，OsAAP8在氨基酸转运活跃的根和节中也有较高表达，暗示其在氨基酸分配的多个过程中都发挥作用。我们构建了OsAAP8的突变体、过表达以及启动子融合GUS的遗传材料，后续将结合这些材料从生理生化和分子生物学角度，解析OsAAP8介导氨基酸分配利用的分子机制，阐明其调控水稻氮素营养利用的具体过程，为进行氮素营养高效的水稻改良提供重要的理论依据和基因资源。

氨基酸态氮是水稻体内转运利用的主要氮素形态之一，经木质部和韧皮部的氨基酸态氮的长途转运是其在不同部位之间进行分配和积累的基础，其中叶片储存氨基酸的再分配以及根向地上部的氨基酸转运是维持植物氮素供应的两个重要过程。我们依据基因表达特征筛选到了参与水稻氨基酸长途转运的因子OsAAP8。基因表达分析表明OsAAP8在叶片氨基酸再利用的关键期被诱导表达，在氨基酸转运活跃的根和节中也有较高表达；OsAAP8启动子驱动GUS表达的转基因材料的染色结果表明OsAAP8主要在维管组织表达；亚细胞定位分析结果发现OsAAP8主要定位于细胞质膜；酵母互补实验和15N标记氨基酸的吸收实验表明OsAAP8具有将胞外氨基酸吸收至体内的能力；综合以上结果，暗示OsAAP8可能参与氨基酸的长途转运，介导水稻体内多个部位的氨基酸利用过程。我们构建获得了OsAAP8的突变体和过量表达材料，表型分析结果发现osaap8突变体中氨基酸向地上部的转运增强，植株生长被促进，表现出株高增高、分蘖增多和产量增加的表型，过量表达材料则表现出相反的表型。氮素指标测定的结果表明，OsAAP8在叶片氨基酸的再利用过程中的贡献不大，而可能主要在根向地上部的氨基酸长途转运中起作用。序列变异位点分析发现，OsAAP8启动子区域的序列在水稻自然群体材料中存在变异，且多个关键的单倍型与分蘖的数目表现出一定的相关性。综合以上结果，该项目的研究表明OsAAP8可能参与水稻根部氨基酸态氮经由木质部的卸载过程，继而影响氨基酸态氮向地上部的运输，并负调控水稻的生长、分蘖和产量。该研究为进行氮素营养高效的水稻改良提供了重要的理论依据和基因资源。