中国南方红壤中活性铝严重制约着紫花苜蓿在南方的生长。申请人成功克隆四个紫花苜蓿LEA基因。转拟南芥证明它们能显著提高拟南芥耐铝性能。本课题拟进行如下实验：①通过LEA启动子连接报告基因转化拟南芥等方法分析LEA对铝胁迫响应的机制；②通过将LEA基因过表达和RNAi干扰的载体分别转入苜蓿单株系，检测铝胁迫下转基因植株生长和各项生理指标，分析细胞膜的完整性、ROS组分与抗氧化系统的变化、光合的变化和铝离子分布的变化，明确LEA蛋白在铝胁迫下对细胞活性的生理保护功能；③利用体外实验体系，通过结合蛋白质组学、生物化学、生物物理学、蛋白结构分析等方法，分析LEA蛋白对蛋白、膜的保护作用，对金属离子的亲和性，和消除氧自由基的能力。同时进一步分析LEA蛋白作用过程中其二级结构发生的变化，明确LEA蛋白的保护作用机制。课题的结果将揭示LEA蛋白保护植物细胞免受铝毒害的作用原因，并丰富LEA蛋白的理论。

Aluminum in red soil in South China seriously restricts the growth of alfalfa. The genes expression of alfalfa under aluminum stress were analyzed with Medicago truncatula chip, and multiple LEA genes were found responding to aluminum stress. Four LEA genes were cloned successfully from alfalfa. Analysis of transgenic arabidopsis thaliana showed that these genes could significantly improve the aluminum tolerance of arabidopsis. This project intends to explore the mechanism of LEA expression regulation by promoter analysis. We plan to clear the physiological protection function of LEA on cell activity under Aluminum Stress by transforming over expression vector and RNAi vector to alfalfa, analyzing cell membrane integrity, ROS components and antioxidant system changes, photosynthetic change and aluminum ion distribution of the genetically modified alfalfa. We plan to analyze the protective effect of LEA on the membrane protein, the affinity for metal ions, and the ability to eliminate oxygen free radicals by in vitro system. At the same time, the changes of the secondary structure of LEA are planned to be further analyzed, and the protective mechanism of LEA protein will be clearly defined. The results will reveal the mechanism of LEA protecting plant cells from aluminum toxicity. These discoveries will enrich the theory of LEA protein.