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自称为顶端分生组织(SAM)的特殊器官发生结构。SAM显示出独特的结构组织，以组织带状和细胞分层为标志。植物SAM的结构与其功能有关，因此新的叶片从SAM的外围区域开始，而中央区域补充在器官发生过程中丢失的新的分生组织细胞。这些结构和功能上不同的SAM区带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实现的。突破性技术的出现使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，它可以从固定在玻片上的固定植物组织中的特定细胞类型中分离出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，这使得可以同时检查15,000到30,000个基因的表达谱。玉米营养分生组织的大小相对较大，大约有250个分生组织细胞被招募到初始的玉米叶片中，这使得这种植物特别适合这个实验系统。激光捕获显微切割/微阵列技术将用于从玉米分生组织和新形成的叶原基的特定区域捕获细胞，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玉米组织转录积累分析(RT-PCR、RNA凝胶杂交和原位杂交)来验证。这些实验将显微解剖分生组织和叶原基中的基因表达模式，并将为植物发育的机制提供新的见解。这项研究项目将产生以下一套交付成果：1.将优化和改进LCM，以用于玉米SAMS。信息将在执行支助股的项目网站和出版物上提供。SAM中全球基因表达的微阵列图谱将存放在公共网站maizeGDB中。3.根据需要，将在项目第二年开始开发富含分生组织的cDNA文库中的EST集合。如果开发出来，所有的EST序列都将保存在Genbank和maizeGDB.4中。学生从其他植物中挖掘已知基因的玉米等价物所产生的序列将直接发送到Genbank和maizeGDB。将通过三种方式加强推广和培训：1.杜鲁门州立大学的本科生将从事尖端基因组学研究。2.皮斯坎隆将在当地招收少数族裔学生的高中做客座演讲。3.佐治亚大学和CSHL Dolan DNA学习中心(DNALC)将对少数族裔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.