Genetic and Molecular Analysis of the Maize kn1 Gene

玉米 kn1 基因的遗传和分子分析

• 批准号: 9727611
• 负责人: Sarah Hake
• 金额: $ 40万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9727611&HistoricalAwards=false
• 关键词: Genetic; Molecular; Analysis; Maize; kn1

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.

9727611始终芽分生组织是负责芽器官发生的自组织中心。 它们在维持不确定细胞的群体的同时，为侧向器官贡献初始细胞。打结1（KN1）编码同源域蛋白TH在芽分生组织中大量表达，并且在叶片中不存在。 分生组织中KN1的下调似乎可以预测下一个叶子将在哪里形成。 KN1在启动分生组织部位的地位生成期间表达。 表达持续到花序，分支和花卉分生组织中，但随着心皮的开始而消失。 KN1玉米功能突变体的丧失在分生组织中有缺陷；从花序形成较少的分支，但额外的确定器官，例如叶子和皮层形成。 KN1在烟草andarabidopsis中的异位表达会引起许多变化，包括非常矮的植物和带有异位芽的叶叶。 从这些表达研究，功能表型的增益和丧失中，我们得出结论，KN1在分生组织中起着重要作用。 提出了一系列实验，以了解KN1的调节和功能，其长期目标是了解射击分生组织的功能。 KN1表达仅限于分生组织的特定域的机制将确定已将构建体放入转基因玉米中，该玉米包含KN1启动子的3KB和KN1 3'端驱动GUS。 四个独立事件的表达揭示了非特异性，无处不在的表达，而不是原位分析所见的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表达的空间调节中的作用。 通过检查35S中的KN1 RNA表达模式：KN1或UBI：KN1转基因植物，将探索转录后调节的程度。 KN1启动子构建体将用于确定KN1是否调节其自身转录。 为了鉴定与K N1相互作用的基因，将进行遗传筛选，以识别增强或抑制功能表型丧失的突变。 酵母两个混合系统将被用来识别其他相互作用的组件，并测试Zag1（玉米agamous同源物）是否与KN1相互作用，因为雌花的功能表型的丧失在Zag1和KN1中相似。 为了识别KN1的下游靶标，构建了诱导系统并转化为拟南芥。 KN1将在有限的时间段内诱导，并从诱导和未诱导的植物中分离出RNA，以比较使用差分显示和PCR-Substraction克隆的转录本种群。 然后，可以识别玉米同源物。 该建议的研究目标，了解KN1受调节的机制，发现与KN1相互作用的蛋白质以及确定潜在目标的蛋白质，将共同增强我们对KN1的理解，从而增加我们对分生组织功能的了解。