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

小行星9630604 结合分子遗传学和生物化学的方法正在被用来研究天冬氨酸转氨酶（AspAT）的模式遗传植物拟南芥中的不同同工酶的功能。 一个主要的目标是确定在体内的作用所发挥的个人AspAT同工酶定位在细胞质，叶绿体，线粒体或过氧化物酶体。 拟南芥突变体中的两个主要的AspAT同工酶（胞质AAT 2或叶绿体AAT 3）的缺陷已被分离和部分特征。将使用显性阴性方法产生在其他“次要”AspAT同工酶（线粒体AAT 1和过氧化物酶体AAT）中有缺陷的另外的拟南芥突变体。将对每种AAT同工酶缺陷的拟南芥突变体进行生理和代谢分析，以确定“缺失”同工酶的体内作用。除了这些遗传学研究外，编码每种AspAT同工酶的克隆基因也在研究中。RFLP作图已被用于确定ASP克隆基因与同工酶突变共分离。 初步结果表明，突变体是在胞质AAT 2的ASP 2基因的胞质AAT 2的缺陷受到影响。 aat 2突变体的表型分析表明，ASP 2编码一个关键的同工酶，控制氮同化成用于细胞间氮运输的天冬氨酸。 基因表达研究还表明，ASP 2起着关键的调节作用，因为其表达受光和/或代谢控制的调节。该项目的具体目的是：1）通过体外细胞器摄取证明ASP 1和ASP 3编码AspAT的线粒体和过氧化物酶体形式。 2)通过原位杂交和/或使用启动子-GUS融合来确定每个ASP基因的细胞特异性表达模式。 3)阐明ASP 2的代谢调控机制。 4)绘制所有ASP基因图谱。 5)确定叶绿体AAT 3中的aat 3突变缺陷是否在ASP结构或调节基因中。 6)定义每个aat 2和aat 3突变体的分子损伤。 7)分离aat 2、aat 3的缺失等位基因和/或调节突变体。 8)使用反义或显性负转基因技术在线粒体AAT 1或过氧化物酶体AAT中产生突变体。9)对aat突变体进行详细的鉴定，包括遗传、生理和生化测试。10)创建异位表达特定ASP基因的转基因植物，这些基因通过突变体分析被定义为关键基因。 本项目是一个分子遗传学分析，主要研究与高等植物氮代谢有关的天冬氨酸氨基转移酶（AspAT）家族。 AspAT的功能是：1）将同化的氮转化为天冬氨酸，用于细胞间氮运输到发育器官（例如种子），2）在无数转氨反应中充当催化剂，为含氮化合物的合成提供氮，3）维持有机氮和碳之间的平衡，4）在亚细胞区室之间穿梭还原当量。这些在拟南芥中的分子遗传学研究将有助于确定哪些特定的AspAT同工酶参与上述过程。 此外，ASP基因在转基因植物中的工程改造具有改善转基因作物中氮利用的潜力。最后，这些研究也将作为一个模型，在其中剖析步骤在其他复杂的多同工酶途径的植物。 ***

9630604 Coruzzi A combined molecular-genetic and biochemical approach is being used to study the function of distinct isoenzymes of aspartate aminotransferase (AspAT) in the model genetic plant Arabidopsis thaliana. A major goal is to identify the in vivo role played by individual AspAT isoenzymes localized in the cytosol, chloroplast, mitochondria or peroxisome. Arabidopsis mutants deficient in either of the two major AspAT isoenzymes (cytosolic AAT2 or chloroplastic AAT3) have been isolated and partially characterized. Additional Arabidopsis mutants defective in the other "minor" AspAT isoenzymes, (mitochondrial AATl and peroxisomal AAT) will be created using a dominant-negative approach. Arabidopsis mutants defective in each AAT isoenzyme will be subject to physiological and metabolic analysis to define the in vivo role of the "missing" isoenzyme. In addition to these genetic studies, the cloned genes encoding each AspAT isoenzyme are also under investigation. RFLP mapping has been used to determine which ASP cloned genes co-segregate with an isoenzyme mutation. Preliminary results demonstrate that the mutants are deficient in cytosolic AAT2 are affected in the ASP2 gene for cytosolic AAT2. The phenotypic analysis of the aat2 mutant suggests that ASP2 encodes a key isoenzyme controlling the assimilation of nitrogen into aspartate used for intercellular nitrogen transport. Gene expression studies have also shown that ASP2 plays a key regulatory role as its expression is regulated by light and/or metabolic control. The specific aims of this project are to: 1) Demonstrate by in vitro organelle uptake that ASPl and ASP3 encode mitochondrial and peroxisomal forms of AspAT. 2) Determine the cell-specific expression pattern of each ASP gene by in situ hybridization and/or by using promoter-GUS fusions. 3) Elucidate the mechanism of metabolic regulation for ASP2. 4) Map all the ASP genes. 5) Determine whether the aat3 mutation deficient in chloroplast AAT3 is in an ASP structural or re gulatory gene. 6) Define the molecular lesion in each aat2 and aat3 mutant. 7) Isolate deletion alleles of aat2, aat3 and/or regulatory mutants. 8) Create mutants in mitochondrial AATl or peroxisomal AAT using anti-sense or dominant-negative transgenic technology. 9) Perform a detailed characterization of the aat mutants including genetic, physiological and biochemical tests. 10) Create transgenic plants which ectopically express specific ASP genes defined to be key by mutant analysis. %%% This project is a molecular genetic analysis that focuses on an important family of related enzymes, aspartate aminotransferase, (AspAT) involved in nitrogen metabolism in higher plants. AspAT functions to 1) convert assimilated nitrogen into aspartate for intercellular nitrogen transport to developing organs (e.g. seed), 2) act as the catalyst in innumerable transamination reactions to donate nitrogen for the synthesis of nitrogen containing compounds, 3) maintain a balance between organic nitrogen and carbon, 4) shuttle reducing equivalents between subcellular compartments. These molecular-genetic studies in Arabidopsis will help define which particular AspAT isoenzyme is involved in each of the above processes. Moreover, the engineering of the ASP genes in transgenic plants has potential for improving nitrogen-use in transgenic crop plants. Finally, these studies will also serve as a model for ways in which to dissect steps in other complex multi-isoenzyme pathways in plants. ***