Functional Genomics of Endosperm Development in Maize

玉米种子胚乳是植物生物学中最重要的结构之一。 该项目的目标是全面的遗传解剖胚乳发育和代谢的分子机制。分析破坏胚乳的突变将允许鉴定控制胚乳发育的基因。 对这些突变体的分子分析将反过来导致在相同或相互作用的途径中起作用的其他基因。识别这一关键的初始基因组的方法可以被视为通过一系列筛选来传递所有玉米基因，每个筛选通过不同的标准来过滤基因。 最后鉴定出一大批赋予胚乳表型的克隆基因。1.筛选一是表型。 原则上，所有在胚乳中具有可见表型的基因都可以通过转座子诱变来标记。 可能需要至少2000个独立突变以确保标记所有胚乳突变体（约300个基因）。 在佛罗里达已经建立了一个足够大的Mutator转座子标记群体，以包含2000个可见的胚乳突变体。 将筛选该群体以鉴定一组富含胚乳基因中的突变体插入的玉米品系。筛选二是胚乳表达。我们的目标是确定标记的基因，其野生型转录本存在于一个大的胚乳cDNA微阵列的子集。 20，000个新的胚乳cDNA将被测序并用于构建微阵列。 通过PCR扩增突变株系中突变体插入位点侧翼的DNA。 通过用来自合并的DNA样品的PCR产物探测微阵列，可以将阵列中代表的基因中的插入追踪到单个玉米品系。 cDNA命中将包括突变基因的候选者，加上与表型无关的偶然基因敲除。 与相同cDNA杂交的独立突变体定义杂交组。 3. 屏幕三是互补测试。 将对每个杂交组内的突变株系进行遗传等位性系统检测。 找到同一基因的独立突变等位基因将确认突变基因克隆的身份。4. 筛选四是生物信息学和功能分析。突变基因序列和表型将用于推断功能并指导所选基因的实验分析。 参与的实验室有互补的兴趣，涵盖胚乳发育的基本过程。参与者：Donald R.麦卡蒂，PI，U。Joachim Messing，联合PI，RutgersBrian Larkins，联合PI，U。来自亚利桑那州的菲利普·贝克拉夫特，爱荷华州州立大学的合作PI。Koch，Co-PI，U.佛罗里达州。Curtis Hannah，Co，PI，U.来自佛罗里达

AbstractThe endosperm of the maize seed is one the most economically important and scientifically interesting structures in plant biology. The goal of this project is comprehensive genetic dissection of the molecular mechanisms underlying endosperm development and metabolism. Analysis of mutations that disrupt the endosperm will allow identification of genes that control endosperm development. Molecular analysis of such mutants will in turn lead to other genes that function in the same or interacting pathways. The approach to identifying this critical initial set of genes can be viewed as passing all maize genes through a series of screens, each filtering genes by a different criterion. In the end a large set of cloned genes that confer endosperm phenotypes are identified. 1. Screen one is phenotype. In principle, all genes that have visible phenotypes in endosperm can be tagged by transposon mutagenesis. At least 2000 independent mutations may be needed to ensure tagging of all endosperm mutants (~300 genes). A Mutator transposon tagging population large enough to contain the 2000 visible endosperm mutants has been created at Florida. This population will be screened to identify a set of maize lines enriched for Mutator insertions in endosperm genes.2. Screen two is endosperm expression. The goal is to identify the subset of tagged genes whose wildtype transcripts are present in a large endosperm cDNA microarray. 20,000 new endosperm cDNA's will be sequenced and used to construct the microarray. DNA flanking Mutator insertion sites in the mutant lines is amplified by PCR. By probing the microarray with PCR products from pooled DNA samples, insertions that are in genes represented in the array can be traced to individual maize lines. cDNA hits will include candidates for the mutant gene, plus incidental gene knockouts not associated with phenotypes. Independent mutants that hybridize to the same cDNA's define a hybridization group. 3. Screen three is complementation testing. Mutant lines within each hybridization group will be systematically tested for genetic allelism. Finding independent mutant alleles of the same gene will confirm the identity of clones of mutated genes. 4. Screen four is bioinformatics and functional analysis. The mutant gene sequence and phenotype will be used to infer function and guide experimental analysis of selected genes. The participating labs have complementary interests covering the essential processes underlying endosperm development. Participants:Donald R. McCarty, PI, U. of FloridaJoachim Messing, Co-PI, RutgersBrian Larkins, Co-PI, U. of ArizonaPhilip Becraft, Co-PI, Iowa State UniversityKaren E. Koch, Co-PI, U. of FloridaL. Curtis Hannah, Co,PI, U. of Florida