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是由称为茎尖分生组织（sam）的特殊器官形成的。SAM具有独特的结构组织，以组织分带和细胞分层为标志。植物SAM的结构与其功能相关，例如，SAM的外围区产生新叶，中心区补充器官发生过程中丢失的新的分生组织细胞。这些结构和功能上截然不同的SAM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。突破性技术的出现使得鉴定和分析玉米分生组织功能所需的基因成为可能。激光解剖显微镜（LCM）是一种强大的技术，可以从固定在载玻片上的固定植物组织中的特定细胞类型中分离RNA。从1,000-10,000个细胞中收集的RNA足以用于基因表达的微阵列分析，这允许同时检查15,000至30,000个基因的表达谱。玉米营养分生组织的大小相对较大，大约250个分生组织细胞被招募到早期的玉米叶片中，使得这种植物特别适合这个实验系统。激光捕获显微解剖/微阵列技术将用于从玉米分生组织的特定区域和新形成的叶片原基中捕获细胞，用于全球基因表达的比较分析。候选基因的差异表达模式将通过更传统的分析（RT-PCR， RNA凝胶杂交和原位杂交）来验证玉米组织中转录物积累。这些实验将微观解剖分生组织和叶原基的基因表达模式，并将为植物发育机制提供新的见解。本研究项目将产生以下一组可交付成果：LCM将在玉米地对空导弹上得到优化和改进。信息将在国际滑联的项目网站和出版物上提供。SAM中基因表达的微阵列图谱将被存放在公共网站maizeGDB上。3. 根据需要，从分生组织富集cDNA文库中收集EST将在项目的第二年开始开发。如果开发成功，所有EST序列将存入Genbank和maizeGDB.4。学生们从其他植物中寻找与玉米相同的已知基因所产生的序列将直接发送到Genbank和maizeGDB。将以三种方式加强外联和培训：杜鲁门州立大学的本科生将参与最前沿的基因组学研究。2. PI Scanlon将在当地以少数民族学生为主的高中进行客座讲座。3. 佐治亚大学和CSHL多兰DNA学习中心（dalc）将对来自少数民族高中和大学的教师进行植物基因组学和生物信息学方面的培训。

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.