A Molecular-Genetic Study of Aspartate Aminotransferase Genes/Isoenzymes in Arabidopsis Thaliana

一种结合分子遗传学的方法正在被用来研究 编码天冬氨酸多种同工酶的基因 氨基转移酶（AspAT）是植物体内氮素代谢的关键酶 新陈代谢. 拟南芥AspAT基因及其缺失突变体 thaliana正在被描述。 目标是确定 每种AspAT同工酶在代谢中的功能， 确定多种AspAT同工酶是否表现不同或 利用分子生物学和遗传学的方法。 的 所用的分子技术将确定 每个单独的AspAT基因与细胞类型，发育， 和光。 这些分子分析结合了 分析特定同工酶中无效突变的影响。 因为AspAT参与了植物中氮的移动， 这些结果对提高植物氮素利用率具有重要意义。 已分离出四种不同的AspAT cDNA。 表征 全长cDNA克隆将使得能够确定 编码的AspAT多肽的一级结构和体外 细胞器摄取研究将确定哪些cDNA编码 叶绿体、线粒体和胞质形式的AspAT。 的 将监测每个AspAT基因的表达模式， 结果用于开发一个关于功能的模型， 编码的同工酶。 每个AspAT基因的启动子元件将是 与报告基因（GUS）融合并导入转基因植物中， 植物 GUS活性的组织化学分析将建立 细胞类型，其中每个AspAT基因表达。 这些信息 将提供对每个基因功能的深入了解。 对每种AspAT生理功能的终极测试 体内同工酶将通过监测生长来确定 在一个或多个方面特异性缺陷的植物的表型 Aspat的形式。 到目前为止，三个假定的AspAT突变体已经 在筛选的750株M2植物中鉴定出了。 遗传杂交 突变体之间的差异将确定不同基因座的数量 影响。 将使用以下方法通过RFLP鉴定结构突变体： 四个克隆的AspAT基因。 AspAT的功能互补 具有克隆的AspAT基因的突变体将把病变限定为 特别是AspAT基因。 %%% 天冬氨酸氨基转移酶是高等植物中的一种关键酶， 在碳和氮骨架之间穿梭 细胞器和细胞之间。 AspAT的生物化学已经被 被多种同工酶的存在所迷惑 我们采取了一 用分子遗传学方法克隆这四种不同的基因 在植物（拟南芥）中编码AspAT。 另外我们 已经开发出一种技术来识别突变植物， 在四种主要的AspAT同工酶中的一种中有特异性缺陷。 克隆的AspAT基因将用于研究结构， 功能和植物中AspAT每种独特形式的调节。 在特定的AspAT同工酶中特异性缺陷的突变体 将对受影响的基因、生长情况、 表型和代谢产物谱，以确定每种 AspAT酶在植物氮代谢中起重要作用。 这些实验 将定义每种AspAT酶的功能， 天冬氨酸在植物生长中的生物合成，也可以作为一个模型 分子遗传学是如何被用来理解 关于植物代谢的问题。

A combined molecular-genetic approach is being used to study the genes encoding the multiple isoenzymes of aspartate aminotransferase (AspAT), a key enzyme in plant nitrogen metabolism. AspAT genes and AspAT deficient mutants of Arabidopsis thaliana are being characterized. The objectives are to determine the function that each AspAT isoenzyme serves in metabolism, and to determine whether the multiple AspAT isoenzymes perform distinct or overlapping roles using molecular biology and genetics. The molecular techniques used will define the expression pattern of each individual AspAT gene with regard to cell-type, development, and light. These molecular analyses combined with the ability to analyze the effects of a null mutation in a particular isoenzyme. Because AspAT is involved in mobilizing nitrogen within a plant, these findings have significance for improving plant nitrogen use. Four distinct AspAT cDNAs have been isolated. Characterization of full length cDNA clones will enable the determination of the primary structure of the encoded AspAT polypeptide and in vitro organellar uptake studies will establish which cDNAs encode chloroplast, mitochondrial, and cytosolic forms of AspAT. The expression pattern of each AspAT gene will be monitored and the results used to develop a model concerning the function of the encoded isoenzyme. Promoter elements for each AspAT gene will be fused to a reporter gene (GUS) and introduced into transgenic plants. Histochemical analysis for GUS activity will establish the cell-type in which each AspAT gene is expressed. This information will provide insight into the function of each gene. The ultimate test of the physiological function of each AspAT isoenzyme in vivo will be determined by monitoring the growth phenotype of plants which are specifically defective in one or more of the forms of AspAT. To date, three putative AspAT mutants have been identified out of 750 M2 plants screened. Genetic crosses between mutants will establish the number of different loci affected. Structural mutants will be identified by RFLP using the four cloned AspAT genes. Functional complementation of the AspAT mutants with the cloned AspAT genes will delimit the lesion to a particular AspAT gene. %%% Aspartate amino transferase is a key enzyme in higher plants which functions to shuttle carbon and nitrogen skeletons between organelles and between cells. The biochemistry of AspAT has been confused by the presence of multiple isoenzymes. We have taken a molecular-genetic approach to clone the four distinct genes encoding AspAT in a plant (arabidopsis thaliana). In addition, we have developed a technique to identify mutant plants which are specifically defective in one of the four major AspAT isoenzymes. The cloned AspAT genes will be used to study the structure, function, and regulation of each unique form of AspAT in plants. The mutants specifically defective in a particular AspAT isoenzyme will be evaluated with regard to the affected gene, the growth phenotype, and metabolite profile to determine the role that each AspAT enzyme plays in plant nitrogen metabolism. These experiments will define the function of each AspAT enzyme, the role of aspartate biosynthesis in plant growth, and also serve as a model for how molecular-genetics can be used to understand fundamental questions concerning plant metabolism.