阿拉善荒漠区珍稀泌盐植物长叶红砂离子转运体系的分子机理研究

Soil salinity is the major environmental factor limiting plant growth and productivity. The detrimental effects of high salinity on plants can be observed at the whole-plant level as the death of plants and/or decreases in productivity. Salinized land worldwide could reach 10% of the land area. The reduction of grain and the irrigated land area together with the other economic losses due to salinity is huge. Therefore, administrating and utilizing the large area of salinized land is extremely urgent. Plant genetic engineering is recognized as an economic and effective way in improving the capacity of plant salt tolerance, developing and utilizing salinization and desertification land. Finding out the salt-resistant determinants is the premise and key to do such jobs. Ion transport is one of the most significant adaptation mechanisms when plants grow under salt-stress environment.Studies on the key elements of ion homeostasis including Na+/H+ antiporter, K+ pumps, plasma membrane and tonoplast H+-ATPase shows that they all play important role in adjusting ion balance of plant cell under salinity condition. In recent years, research of ion transport mechanisms between Arabidopsis thaliana and Thellungiella halophila show that they are different in Na+ absorption and efflux, revealing that there probably exist novel ion transporter and regulatory element in the halophytes.Clearly, it is obvious that studying the salt tolerance mechanisms of halophyte species to find the newly ion homeostasis related genes is of great significance. The Eastern Alxa-Western Ordos area is one of the eight biodiversity centers in China. This area retained many rare and endangered plants that have strongly resistance characteristics and offers a huge natural library of stress-resistant genes. Reaumuria trigyna is a recretohalophyte, also known as a "living fossil" that only found in China. The unique multicellular salt gland of R. trigyna has the property of ion selection efflux. It can excrete more Na+, Cl-, and SO42- apparently higher than any other kind of ions. Therefore, it is thought that there are special ion transporters and regulation mechanisms in this plant. Based on the transcriptome sequencing data, this project will 1)identify and functional analyze the novel ion transporter and regulatory element of R. trigyna by using of molecular biology and related methods 2)set the ion transport model of this plant in response to salt stress 3)compare the functional differences of the certain ion regulated gene derived from R. trigyna and its homologous gene from A. thaliana and T.halophile 4)transform R. trigyna ion adjusting determinants into Medicago sativa and evaluate the salt resistant ability of transgenic plants. The implementation of the project will provide new thinking in studying salt tolerance mechanisms of wild native plants and will laid a foundation for the cultivation of new varieties of salt tolerance plants used for the salinity soil improvement.

土地盐渍化是植物生长的主要环境限制因子,研究植物耐盐机理,运用基因工程手段提高植物耐盐性是治理和利用大面积盐渍化土地经济有效的途径。盐胁迫条件下，植物通过膜系统表面的离子转运蛋白完成过量盐离子的外排及胞内区隔化，近年对模式植物拟南芥和盐芥的研究表明：二者的盐胁迫离子转运响应机制存在差异，揭示了研究野生盐生植物具有发现耐盐决定因子及信号通路的更大潜力。本项目以阿拉善荒漠区特有珍稀泌盐植物长叶红砂为材料，在对高通量转录组数据分析的基础上，克隆该植物质膜和液泡膜离子转运通路相关基因，对其进行表达模式分析、实施这些基因和拟南芥、盐芥中的同源基因与相应拟南芥突变体的功能互补实验并在耐盐牧草紫花苜蓿中进行过量表达，结果将为构建长叶红砂响应盐胁迫的离子转运模型，阐述不同离子转运蛋白在离子稳态重建中的作用，比较该植物与拟南芥、盐芥在离子转运特性方面的异同，发掘用于作物改良的优质耐盐工程基因奠定基础。

长叶红砂作为古老且分布狭窄的珍稀泌盐植物，对盐渍化荒漠环境具有极强的适应性。本项目以长叶红砂为研究对象，基于转录组数据，筛选并克隆了长叶红砂中9个在盐胁迫条件下差异表达基因，包括4个不同类型的离子转运蛋白基因（RtNHX1、RtVP1、RtHKT1和RtKCO1），2个WRKY转录因子（RtWRKY1和RtWRKY23）和3个与黄酮合成相关的基因（RtF3H1、RtF3H2 和RtLDOX1），并对其编码的蛋白质的分子特征、表达特性以及功能进行了分析，探讨在盐胁迫条件下，植物如何通过离子转运蛋白的协同作用重建体内离子稳态平衡、黄酮类次生代谢物质的积累对提高植物耐盐性的作用、WRKY转录因子对植物耐受非生物胁迫的调控作用等问题，为揭示目的基因的功能及其在植物耐盐性遗传改良中的应用价值奠定基础。结果显示：1. RtNHX1、RtVP1、RtHKT1和RtKCO1基因通过调控转基因植物体内的离子平衡，降低Na+/K+、提高渗透调节物质积累和抗氧化酶活性，进而增强了转基因植物的盐耐受性。2.离子转运蛋白的协同作用在植物Na+-K+离子稳态平衡的重建中发挥了重要作用。在外部高钠环境下，液泡膜RtNHX1蛋白能够将细胞中多余的Na+和K+同时区隔化到液泡中，同时液泡膜RtVP1能够将胞质中的H+泵入液泡内建立质子电化学势为RtNHX1提供动力来源，液泡膜上的RtNHX1和RtVP1蛋白均能使转基因拟南芥叶片中保持较低的Na+离子和较高的K+离子含量；质膜上的RtHKT1蛋白能够促进转基因拟南芥叶片钾离子的积累，阻止钠离子的吸收；而质膜RtKCO1蛋白能将转基因拟南芥叶片中过多的Na+和K+同时排出体外，且钠离子排出速率大于钾离子；从而导致转RtNHX1、RtVP1、RtHKT1和RtKCO1基因拟南芥中保持较低的Na+/K+，使植物对外部高钠环境更加耐受。3. 转RtHKT1基因拟南芥的盐耐受性依赖于外部适当浓度的K+；RtHKT1和RtKCO1蛋白的离子转运特性依赖于外部Na+-K+环境。4. RtWRKY1和RtWRKY23能够通过调节植株在盐胁迫条件下的抗氧化及渗透调节能力且提高植物的耐盐性。RtF3H1、RtF3H2和RtLDOX1基因能够提高植物体内的黄酮积累和活性氧清除能力，进而改善转基因拟南芥的盐耐受性。