Functional Analyses of Genes Involved in Meristem Organization and Leaf Initiation

高等植物的所有地上器官最终都来源于被称为茎顶端分生组织（SAMs）的特殊器官发生结构。 SAM具有独特的结构组织，以组织分区和细胞分层为标志。 植物自组装膜的结构与其功能相关，因此新叶从自组装膜的外围区开始，而中心区重新形成在器官发生过程中丢失的新的分生组织细胞。 SAM的这些结构和功能不同区域的建立和维持是通过数千个植物基因的差异表达模式和相互作用来实现的。 突破性的技术已经出现，使玉米分生组织功能所需的基因的鉴定和分析。 激光解剖显微镜（LCM）是一种强大的技术，允许从固定在载玻片上的固定植物组织内的特定细胞类型中分离RNA。 从1，000 - 10，000个细胞收集的RNA足以用于基因表达的微阵列分析，其允许同时检查15，000至30，000个基因的表达谱。 玉米营养分生组织的相对大的尺寸，约250个分生组织细胞被募集到初期玉米叶中，使得该植物特别易于用于该实验系统。 激光捕获显微切割/微阵列技术将被用来捕获玉米分生组织和新形成的叶原基用于全球基因表达的比较分析的特定领域的细胞。 候选基因的差异表达模式将通过玉米组织中转录本积累的更传统的分析（RT-PCR、RNA凝胶印迹杂交和原位杂交）来验证。 这些实验将显微解剖基因表达模式的分生组织和叶原基，并将提供新的见解植物发育的机制。 本研究项目将产生以下一组可交付成果：1。 LCM将进行优化和改进，以用于玉米SAM。有关资料将在支助股的项目网站和出版物中提供。 SAM中全局基因表达的微阵列谱将被存放在公共网站maizeGDB中。3. 根据需要，将在项目的第二年开始从分生组织富集的cDNA文库中收集EST。如果开发出来，所有EST序列将存入Genbank和玉米GDB。 学生们挖掘玉米等同于其他植物已知基因所产生的序列将直接发送到Genbank和maizeGDB。 来自杜鲁门州立大学的本科生将从事尖端基因组学研究。2. PI Scanlon将在当地以少数民族为主的高中进行客座讲座。 3. 格鲁吉亚大学和CSHL多兰DNA学习中心（DNALC）将培训教师从代表性不足的少数民族高中和大学设置在植物基因组学和生物信息学。

All above ground organs of higher plants are ultimately derived from specialized organogenic structures called shoot apical meristems (SAMs). The SAM exhibits distinctive structural organization, marked by tissue zonation and cell layering. The structure of plant SAMs is correlated with their function, such that new leaves are initiated from the peripheral zone of the SAM and the central zone replenishes new meristematic cells that are lost during organogenesis. Establishment and maintenance of these structurally and functionally distinct zones of the SAM is achieved by differential expression patterns and interactions of thousands of plant genes. Breakthrough technologies have emerged that enable the identification and analysis of genes required for meristem function in maize. Laser dissection microscopy (LCM) is a powerful technique that permits the isolation of RNA from specific cell types within fixed plant tissues immobilized on slides. RNA collected from 1,000-10,000 cells is sufficient for use in microarray analyses of gene expression, which permit the simultaneous examination of expression profiles of 15,000 to 30,000 genes. The relatively large size of the maize vegetative meristem, approximately 250 meristematic cells are recruited into the incipient maize leaf, renders this plant especially tractable for this experimental system. The laser-capture microdissection/microarray technique will be used to capture cells from specific domains of the maize meristem and newly formed leaf primordia for use in comparative analyses of global gene expression. The differential expression patterns of candidate genes will be verified by more traditional analyses (RT-PCR, RNA gel blot hybridization and in situ hybridization) of transcript accumulation in maize tissues. These experiments will microdissect gene expression patterns in meristems and leaf primordia, and will provide novel insight into mechanisms of plant development. This research project will generate the following set of deliverables:1. LCM will be optimized and improved for use on maize SAMs. Information will be available on a project web site at ISU and in publications.2. Microarray profiles of global gene expression in the SAM will be deposited in public web sites, maizeGDB. 3. An EST collection from meristem-enriched cDNA library will be developed beginning in the second year of the project, depending on need. If developed, all EST sequences will be deposited in Genbank and in maizeGDB.4. Sequence generated by students mining for maize equivalents to already known genes from other plants will be sent directly to Genbank and maizeGDB.Outreach and training will be enhanced in three ways:1. Undergraduate students from Truman State University will engage in cutting edge genomics research. 2. PI Scanlon will deliver guest lectures at local high schools with predominantly minority enrollment. 3. The University of Georgia and the CSHL Dolan DNA Learning Center (DNALC) will train teachers from underrepresented minority high schools and university settings in plant genomics and bioinformatics.