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

利用分子遗传学和生物化学相结合的方法研究了模式遗传植物拟南芥中不同的天冬氨酸转氨酶（AspAT）同工酶的功能。主要目标是确定定位于细胞质、叶绿体、线粒体或过氧化物酶体中的单个AspAT同工酶在体内所起的作用。已经分离出两种主要的AspAT同工酶（胞质AAT2或叶绿体AAT3）缺乏的拟南芥突变体，并对其进行了部分鉴定。其他“次要”AspAT同工酶（线粒体AATl和过氧化物酶体AAT）缺陷的拟南芥突变体将使用显性阴性方法创建。每个AAT同工酶缺陷的拟南芥突变体将进行生理和代谢分析，以确定“缺失”的同工酶在体内的作用。除了这些遗传研究之外，编码每一种AspAT同工酶的克隆基因也在研究中。RFLP图谱已用于确定哪些ASP克隆基因与同工酶突变共分离。初步结果表明，胞质AAT2缺陷突变体受到胞质AAT2的ASP2基因的影响。对aat2突变体的表型分析表明，ASP2编码一种关键同工酶，控制氮同化成用于细胞间氮运输的天冬氨酸。基因表达研究也表明，ASP2的表达受到光和/或代谢控制的调节，因此它起着关键的调节作用。该项目的具体目标是：1)通过体外细胞器摄取证明ASPl和ASP3编码AspAT的线粒体和过氧化物酶体形式。2)通过原位杂交和/或启动子- gus融合确定每个ASP基因的细胞特异性表达模式。3)阐明ASP2的代谢调控机制。4)绘制所有ASP基因图谱。5)确定叶绿体aat3突变缺陷是否在ASP结构基因或调控基因上。6)确定每个aat2和aat3突变体的分子病变。7)分离aat2、aat3和/或调节突变体的缺失等位基因。8)利用反义或显性阴性转基因技术制造线粒体AATl或过氧化物酶体AAT突变体。9)对aat突变体进行详细的表征，包括基因、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，通过突变体分析确定其为关键基因。本项目是对高等植物中参与氮代谢的一个重要的相关酶家族——天冬氨酸转氨酶（AspAT）进行分子遗传分析。AspAT的功能是：1)将同化的氮转化为天冬氨酸，用于细胞间氮转运到发育器官（如种子）；2)在无数的转氨化反应中充当催化剂，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)在亚细胞间穿梭还原性当量。这些拟南芥的分子遗传学研究将有助于确定哪种特定的AspAT同工酶参与上述每个过程。此外，在转基因植物中进行ASP基因的工程改造，对改善转基因作物的氮素利用具有潜在的意义。最后，这些研究也将为剖析植物中其他复杂的多同工酶途径的步骤提供模型。＊＊＊

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. ***