Molecular Analysis of Salt Tolerance Proteins

土壤盐分是降低植物生长和生产力的主要因素。大多数农作物对盐胁迫高度敏感，盐胁迫往往会因渗透胁迫而导致缺水，并因细胞内钠有毒过量而导致多种生化问题。有些植物品种的耐盐性差别很大。钠/氢（Na+/H+）反向转运蛋白是从植物和酵母细胞内去除盐的膜蛋白。它们去除盐（钠）以换取细胞外部的质子。Na+/H+反向转运蛋白的过表达已显示赋予植物改善的耐盐性，允许它们在更咸的土壤中生长。我们的项目的目的是了解Na+/H+反向转运蛋白是如何工作的，也就是说，更好地了解它们将盐从细胞中移除并允许植物在盐存在的土壤中生长的方式。为此，我们使用酵母模型系统来研究Na+/H+反向转运蛋白的功能。酵母具有Na+/H+反向转运蛋白，其与植物Na+/H+反向转运蛋白非常相似。我们已经制造了一种酵母菌株，其自身的Na+/H+反向转运蛋白被敲除。它不能在含有盐的培养基中生长。植物和酵母Na+/H+逆向转运蛋白相似，可以相互替换并正常发挥功能。我们可以将植物Na+/H+反向转运蛋白返回到酵母或蛋白质的改变形式中。然后，我们可以通过检查酵母是否在含有盐的培养基中生长来测试它是否起作用。基于分析蛋白质序列的计算机程序，我们已经建立了蛋白质的分子模型。为了确认蛋白质的哪一部分是重要的，我们正在植物和酵母蛋白质中进行突变，这是基于分子模型。然后，我们将突变蛋白返回敲除酵母并测试盐耐受性是否恢复。这使我们能够确定蛋白质的哪些部分是重要的。该项目的另一部分是试图获得蛋白质的外观如何决定其形状或细胞膜中的拓扑结构的图片。为此，我们制作了一种改变的Na+/H+反向转运蛋白，该蛋白去除了所有的半胱氨酸氨基酸残基，但仍然具有功能。我们将在蛋白质的特定位置重新引入单个半胱氨酸氨基酸，并在我们的敲除酵母中表达。然后，我们将使用与半胱氨酸氨基酸反应的特定化合物。我们可以通过这样的方式来确定氨基酸是在细胞外，细胞内还是细胞膜内。我们将根据我们的计算机模型在蛋白质的特定点上重复进行。然后，我们将能够建立一个经过验证的蛋白质图片。该项目的另一部分是制造一种过度活跃的耐盐蛋白。我们将随机诱变几种Na+/H+反向转运蛋白。然后我们将它们重新引入酵母中，并在含盐量高的培养基中培养酵母。然后，我们可以分析允许超级耐盐的突变。在不同的Na+/H+反向转运蛋白中整理这些信息可能使我们能够设计出超级耐盐蛋白，这些蛋白可能有助于重新引入植物，使它们更能够在含盐土壤中生长。总的来说，该项目将帮助我们了解蛋白质的工作原理，使我们能够更好地设计可用于提高植物耐盐性的蛋白质。

Soil salinity is a major factor in reducing plant growth and productivity. Most crop plants are highly sensitive to salt stress that tends to cause a water deficit due to osmotic stress, and that causes several biochemical problems due to toxic excess intracellular sodium. The degree of salt tolerance of some plant species varies greatly. Sodium/Hydrogen (Na+/H+) antiporters are membrane proteins that remove salt from inside the cells of plants and yeast. They remove salt, (sodium) in exchange for a proton that is on the outside of the cell. Overexpression of Na+/H+ antiporters has been shown to confer improved salt tolerance in plants allowing them to grow in saltier soils. The purpose of our project is to understand how Na+/H+ antiporters work, that is, to better understand the way that they remove salt out of cells and allow plants to grow in soil with salt present. To do this we are using a model system of yeast to study how Na+/H+ antiporters function. Yeast have a Na+/H+ antiporter that is very similar to plant Na+/H+ antiporters. We have made a yeast strain that has its own Na+/H+ antiporter knocked out. It doesn’t grow in medium that has external salt. Plant and yeast Na+/H+ antiporter proteins are similar and can replace each other and function normally. We can return a plant Na+/H+ antiporter into the yeast or an altered form of the protein. Then we can test if it is functioning by checking if the yeast grow in medium containing salt. We have made molecular models of the proteins, based on computer programs that analyze the proteins sequence. To confirm which part of the protein is important we are making mutations in the plant and yeast proteins, that are based on the molecular models. We then return the mutant protein back into the knockout yeast and test if salt tolerance is returned. This allows us to determine which parts of the protein are important. Another part of the project is to try to get a picture of how the protein looks determining its shape, or topology in the cell membrane. To do this, we made an altered Na+/H+ antiporter protein that has all its cysteine amino acid residues removed, but is still functional. We will reintroduce single cysteine amino acids in specific places in the protein and express it in our knockout yeast. We will then use specific chemical compounds that react with cysteine amino acids. We can do this in such as way as to determine if the amino acid is on the outside of the cell, on the inside of the cell or within the membrane of the cell. We will do this repeatedly and in specific spots on the protein based on our computer models. We will then be able to build a verified picture of the protein. Another part of the project is to make a hyperactive salt tolerance protein. We will randomly mutagenize several Na+/H+ antiporter proteins. Then we will reintroduce them into the yeast and grow the yeast in high salt containing medium. We can then analyze the mutations that allowed super salt tolerance. Collating this information in different Na+/H+ antiporters may allow us to design super salt tolerance proteins that might be useful to reintroduce back into plants, making them more able to grow in salt containing soil. Overall, the project will help us understand how the protein works, allowing us to better design proteins that can be used to improve salt tolerance in plants.