Role of LEAFY in Determining Floral Identity

花发育的中心步骤是一组未分化的细胞决定形成花的命运。绿叶基因在这一过程中起着关键作用，其活性是促进花高于茎的发育所必需的。本研究从拟南芥中克隆了LEAFY基因，发现其编码一种新型核蛋白，这是已知最早在初生花原基中激活的基因产物。他建议通过阐明绿叶蛋白如何在分子水平上发挥其功能，并通过剖析绿叶启动子的顺式调控元件来详细研究绿叶如何控制花的命运。由于LEAFY编码一种核蛋白，因此它是否编码一种新型的转录调节因子将被确定。该蛋白含有两个与已知转录调控因子的转录激活域相似的结构域。这些结构域的体内活性将通过向转基因植物中引入叶类基因的体外工程缺失变体，并测试这些结构域是否与叶类基因突变互补来确定。我们将通过体外合成的LEAFY蛋白结合DNA的能力测试来探索其作为一种新型DNA结合蛋白的可能性。此外，叶蛋白与其他已知在花发育中起作用的蛋白的分子相互作用也将被研究。未来，将根据其在体外结合LEAFY蛋白的能力分离出新的蛋白。将通过三种策略鉴定介导LEAFY表达时空控制的顺式作用元件：首先，将含有5‘和3’序列组合的构建体以及融合到LEAFY cDNA的内含子序列进行检测，以便在转基因植物中对lfy突变表型进行互补。其次，将snapdragon的LEAFY同源同源物FLORICAULA的启动子序列与LEAFY启动子序列进行比较，以确定co保守的序列元件。第三，5.4 kb片段的各个部分，包括5‘和3’以及内含子序列，将在转基因植物中测试它们对报告基因表达的影响。研究在两个花特异启动子（两个花控制基因APETALA1和AGAMOUS）的控制下，LEAFY的表达是否能完全或部分地补足叶突变体的表型，将对报告基因构建的LEAFY调控序列分析进行补充。这些实验将允许评估在APETALA1和AGAMOUS之前被激活的LEAFY的早期表达是否确实是其体内功能所必需的。这项研究将进一步加深我们对花是如何产生的理解，并将是朝着实现对这一过程的机制理解的最终目标迈出的重要一步。在未来，这将允许定向操纵开花不仅在模型系统，如拟南芥，而且在其他，经济上重要的开花植物。这将改善生殖器官和营养器官之间光同化物质的分配，从而提高作物产量。

A central step in flower development is the decision of a group of undifferentiated cells to adopt a floral fate. A key role in this process is played by the LEAFY gene, whose activity is required to promote floral over shoot development. This investigator has cloned the LEAFY gene from Arabidopsis and shown that it encodes a novel type of nuclear protein, which is the earliest known gene product to be activated in nascent flower primordia. He proposes to study in detail how LEAFY controls floral fate by elucidating how LEAFY protein exerts its function at the molecular level, and by dissecting the cis-regulatory elements of the LEAFY promoter. Since LEAFY encodes a nuclear protein, it will be determined whether it encodes a novel type of transcriptional regulator. The protein contains two domains with similarity to transcriptional activation domains of known transcriptional regulators. The in vivo activity of these domains will be determined by introducing in vitro engineered deletion variants of LEAFY into transgenic plants and testing these constructs for complementation of leafy mutations. The possibility that LEAFY acts as a novel type of DNA-binding protein will be explored by testing in vitro synthesized LEAFY protein for its ability to bind DNA. In addition, the molecular interaction of LEAFY with other proteins known to have a role in floral development will be studied. In the future, new proteins will be isolated based on their ability to bind LEAFY protein in vitro. Cis-acting elements mediating the temporal and spatial control of LEAFY expression will be identified by three strategies: First, constructs containing combinations of 5' and 3' sequences as well as intron sequences fused to a LEAFY cDNA will be tested for complementation of the lfy mutant phenotype in transgenic plants. Second, the promoter sequence of the LEAFY cognate homolog from snapdragon, FLORICAULA, will be compared to that of the LEAFY promoter, in order to identify co nserved sequence elements. Third, various parts of the 5.4 kb fragment, including 5' and 3' as well as intron sequences, will be tested in transgenic plants for their effects on the expression of a reporter gene. The analysis of LEAFY regulatory sequences with reporter gene constructs will be complemented by studying whether expression of LEAFY under the control of two floral specific promoters, those of the two floral control genes APETALA1 and AGAMOUS, can completely or partially complement the leafy mutant phenotype. These experiments will allow for assessing whether the early expression of LEAFY, which is activated before APETALA1 and AGAMOUS, is indeed required for its in vivo function. The proposed research will further our understanding of how flowers are initiated, and will be an important step toward the ultimate goal of achieving a mechanistic understanding of this process. In the future, this will permit for directed manipulation of flowering not only in model systems such as Arabidopsis, but also in other, economically important flowering plants. This should lead to improved partitioning of photoassimilates between reproductive and vegetative organs, and thus to better crop yields.