Genetic and Molecular Analysis of the Maize kn1 Gene

9727611根茎分生组织是负责根茎器官发生的自组织中心。它们为外侧器官提供初始细胞，同时维持不确定的细胞群。knottted1 （kn1）编码一种同源结构域蛋白，该蛋白在茎分生组织中大量表达，而在叶片中不存在。分生组织中kn1的下调似乎预示着下一片叶子将在哪里形成。Kn1在胚胎发生过程中在起始分生组织的位置表达。在花序、分枝和花分生组织中持续表达，但在心皮形成时消失。玉米Kn1功能缺失突变体在分生组织维持方面存在缺陷；较少的分枝从花序形成，但额外确定的器官，如叶和心皮形成。kn1在烟草和拟南芥中的异位表达导致了许多变异，包括非常矮小的植株和带有异位芽的裂叶。从这些表达研究，功能表型的获得和损失，我们得出结论，kn1在分生组织维持中起重要作用。为了了解茎部分生组织的功能，我们提出了一系列的实验来了解kn1的调控和功能。将一个含有3kb的kn1启动子和驱动GUS的kn1 3′末端的构建体放入转基因玉米中，这将决定kn1表达限制在分生组织特定结构域的机制。原位分析显示，四个独立事件的表达显示非特异性，普遍表达而不是分生组织特异性表达。进一步的转基因实验将测试内含子在kn1表达空间调控中的作用。通过检测35S:kn1或UBI:kn1转基因植物中的kn1 RNA表达模式，将探索转录后调控的程度。kn1启动子结构将用于确定kn1是否调节其自身的转录。为了鉴定与kn1相互作用的基因，将进行遗传筛选以鉴定增强或抑制kn1功能表型丧失的突变。酵母双杂交系统将用于鉴定其他相互作用成分，并测试zag1（玉米AGAMOUS同源物）是否与kn1相互作用，因为zag1和kn1的雌花丧失功能表型相似。为了鉴定kn1的下游靶点，我们构建了一个诱导系统，并将其转化为拟南芥。kn1将被诱导一段有限的时间，并从诱导和未诱导的植物中分离RNA，使用差异显示和pcr -减法克隆来比较转录本的群体。鉴定玉米同源物是可能的。本提案的研究目标，了解kn1的调节机制，发现与kn1相互作用的蛋白质，并确定潜在的靶点，将共同增加我们对kn1的理解，从而增加我们对分生组织功能的了解。

9727611 Hake Shoot meristems are self-organizing centers responsible for organogenesis of the shoot. They contribute initial cells for lateral organs while maintaining a population of indeterminate cells. Knotted1 (kn1) encodes a homeodomain protein tht is abundantly expressed in shoot meristems and is absent in leaves. The down-regulation of kn1 in the meristem appears to predict where the next leaf will form. kn1 is expressed during embyrogenesis at the site of the initiating meristem. Expression persists into the inflorescence, branch and floral meristems, but disappears as carpels initiate. kn1 loss of function mutants in maize are defective in meristem maintenance; fewer branches form from the inflorescence but extra determinate organs such as leaves and carpels form. Ectopic expression of kn1 in tobacco andArabidopsis causes a number of alterations including very dwarfed plants and lobed leaves bearing ectopic shoots. From these expression studies, gain and loss of function phenotypes, we conclude that kn1 plays a significant role in meristem maintenance. A series of experiments are proposed to understand the regulation and function of kn1 with the long term goal of understanding how shoot meristems function. The mechanism by which kn1 expression is restricted to specific domains of the meristem will determined A construct has been placed into transgenic maize that contains 3kb of the kn1 promoter and the kn1 3' end driving GUS. The expression of four independent events revealed non-specific, ubiquitous expression rather than meristem-specific expression as seen with in situ analysis. Further transgenic experiments will test the role of the introns in spatial regulation of kn1 expression. The extent of posttranscriptional regulation will be explored by examining kn1 RNA expression patterns in 35S:kn1 or UBI:kn1 transgenic plants. The kn1 promoter construct will be used to determine whether KN1 regulates its own transcription. In order to identify genes that interact with k n1, genetic screens will be carried out to identify mutations that enhance or suppress the kn1 loss of function phenotype. The yeast two hybrid system will be utilized to identify other interacting components and to test whether zag1, the maize AGAMOUS homolog, interacts with kn1 since the loss of function phenotype of the female flower is similar for zag1 and kn1. To identify downstream targets of kn1, an inducible system was constructed and transformed into Arabidopsis. kn1 will be induced for a limited time period and RNA isolated from induced and uninduced plants to compare the populations of transcripts using differential display and PCR-substraction cloning. Identification of the maize homolog is then possible. The research goals of this proposal, understanding the mechanism by which kn1 is regulated, finding proteins that interact with KN1, and identifying potential targets, will collectively increase our understanding of kn1 and thus increase our knowledge of how meristems function.