Functional Analysis of Agamous

这些研究的长期目标是了解控制拟南芥花发育的分子机制。确定了三个具体目标：(1)PI将使用诱导启动子融合分析agamous （AG）功能。重点将放在已经产生的转基因植物上，这些植物携带AG与热休克启动子的融合体。PI将在不同时间进行温度变化实验，并研究这种处理的效果。(2) PI将识别和表征AG结合位点。他在体外鉴定了DNA与野生型AG蛋白的结合。他将使用DNA结合试验来表征他在大肠杆菌中表达的几种AG截断突变蛋白。他还将测试在转基因植物中引起非常有趣的ag样表型的一点突变体。PI还计划测试酵母中的DNA结合，并将使用该系统分析不同的AG靶位点，包括来自AGL1基因的位点。(3) PI将测试可能的AG靶基因，通过体外和体内试验研究AG与AGL1 5'非转录区和AGL1大内含子中潜在AG结合位点的结合。体外鉴定的位点将进一步在酵母和转基因植物中使用报告基因构建进行分析。这些研究将进一步加深我们对AG在花发育过程中的功能的理解，特别是它在调控层次结构中作为基因转录调节剂的作用。* * *

9316194 Ma The long term goal of these studies is to understand the molecular mechanisms controlling flower development in Arabidopsis. Three specific aims are identified: (1) The PI will analyze agamous (AG) function using inducible promoter fusions. The focus will be on already generated transgenic plants that carry fusions of AG with a heatshock promoter. The PI will perform temperature shift experiments at different times and study the effects of such treatment. (2) The PI will identify and characterize AG binding sites. He has characterized in vitro DNA binding with wild-type AG proteins. He will use the DNA binding assays to characterize several AG truncation mutant proteins which he has expressed in E. coli. He will also test one point mutant that causes a very interesting ag-like phenotype in transgenic plants. The PI also plans to test DNA binding in yeast and will use this system to analyze different AG target sites, including those from the AGL1 gene. (3) The PI will test possible AG target genes, using in vitro and in vivo assays to study AG binding to potential AG-binding sites in the 5' AGL1 untranscribed region and in the large AGL1 intron. Sites identified in vitro will be further analyzed in yeast and in transgenic plants using reporter gene constructs. These studies will further our understanding of AG function during flower development, particularly its role as a transcriptional regulator of genes in a regulatory hierarchy. ***