Functional Genomics of Endosperm Development in Maize

摘要玉米种子胚乳是植物生物学中最具经济意义和科学意义的结构之一。该项目的目标是对胚乳发育和代谢的分子机制进行全面的遗传解剖。对破坏胚乳的突变进行分析，将有助于识别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其他基因在相同或相互作用的途径中起作用。鉴定这组关键的初始基因的方法可以看作是通过一系列筛选来筛选所有的玉米基因，每个筛选都用不同的标准来筛选基因。最后，鉴定出一大批赋予胚乳表型的克隆基因。1. 筛选一是表现型。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进行标记。至少需要2000个独立的突变才能确保标记所有胚乳突变（约300个基因）。一个足以容纳2000个可见的胚乳突变体的Mutator转座子标记种群已经在佛罗里达创建。该群体将被筛选以鉴定一组富含胚乳突变体插入的玉米品系。屏幕二是胚乳表达。目的是鉴定野生型转录本存在于大型胚乳cDNA微阵列中的标记基因子集。2万个新的胚乳cDNA序列将被测序并用于构建微阵列。通过PCR扩增突变系中突变体插入位点的DNA。通过用聚合DNA样本的PCR产物探测微阵列，阵列中所代表基因的插入可以追溯到单个玉米品系。cDNA命中将包括突变基因的候选基因，以及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为杂交组。3. 屏幕三是互补测试。每个杂交组内的突变系将被系统地检测遗传等位基因。发现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和指导选定基因的实验分析。参与的实验室具有互补的兴趣，涵盖胚乳发育的基本过程。参与者：Donald R. McCarty， PI, university of florida, joachim Messing, Co-PI, ruters, brian Larkins, Co-PI, university of arizona, philipbecraft, Co-PI, Iowa State university, karen E. Koch, Co-PI, university of florida柯蒂斯汉娜，Co，PI，佛罗里达大学

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