Use of the Transposon Ac as a Gene-searching Engine in the Maize Genome

使用转座子 Ac 作为玉米基因组中的基因搜索引擎

• 批准号: 9813364
• 负责人: Hugo Dooner
• 金额: $ 133.42万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-10-01 至 2001-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9813364&HistoricalAwards=false
• 关键词: Use; Transposon; Ac; Gene; searching

Most of the maize genome (90%) is made up of repetitive DNA, a large fraction of which is methylated. Genes comprise less than 3% of the genome and are found in contiguous stretches of less methylated DNA known as hypomethylated islands. Because of the repetitive DNA content in maize, sequencing the entire maize genome (2.5xl09 bp) is impractical. Some laboratories, mostly in industry, have adopted the strategy of large-scale sequencing of the RNA products transcribed from genes (cDNAs). An attractive alternative is to specifically sequence genes. One can take advantage of the tendency of the maize transposon Activator (Ac) to insert in hypomethylated DNA, the genomic component containing genes, to identify genes as sites into which Ac transposes (tac sites) and, then, to sequence the DNA adjacent to the transposon. An advantage of this approach is that, in addition to a sequence that can be compared to the existing database, it generates an insertion library. The collection of lines carrying Ac at many different locations in the maize genome will enable investigators to screen for subtle mutant phenotypes, particularly after obtaining information on where in the plant the genes are expressed. Many genes are expected to have minor effects and could be missed from a conventional transposon mutagenesis screen designed to identify gross changes in phenotype.Taking advantage of the powerful endosperm genetics of maize, a simple and efficient Ac transposition assay based on the well-studied endosperm markers bz (bronze) and wx (waxy) has been developed. A collection of over 1200 independent Ac transposants has been generated and over one-third of these Ac sites have been mapped relative to the donor locus. In parallel, a panhandle PCR method, originally used in the human genome, has been adapted for the isolation of DNA adjacent to the insertion (tac sites). By sequencing tac sites, insertions have been identified, for example, in genes encoding a sulfur transporter, a MAPKK, and a sesquiterpene synthase.This Ac mobilization scheme allows the isolation of tac sites that are either genetically linked or unlinked to the donor locus. However, because Ac has a strong tendency to transpose to closely linked sites, about one-half of the tac sites are linked to wx on chromosome 9. Clearly, it would be desirable to mobilize Ac from different starting sites in the genome. Toward that end, suitable maize lines that are readily transformable and regenerable are being developed. These lines will be transformed with a construct carrying an Ac element modified to facilitate the isolation of tac DNA. This construct should integrate at random sites in the genome, providing starting platforms for future Ac mobilization.The expected outcomes of this proposal are: (a) The sequence of more than 1000 maize genes or gene fragments. (b) The elucidation of the function of a set of 50 genes based on the sequence of tac sites, the phenotype of Ac insertion mutations, and the pattern of gene expression. (c) The map location of those genes that correspond to unique sequences in the maize genome. (d) The development of transgenic maize lines that will facilitate the future isolation of genes (tac sites) from any location in the genome.

大部分玉米基因组（90%）由重复DNA组成，其中大部分是甲基化的。基因占基因组的不到3%，并且在被称为低甲基化岛的甲基化程度较低的DNA的连续延伸中发现。由于玉米中的重复DNA含量，对整个玉米基因组（2.5xl09bp）进行测序是不切实际的。一些实验室，主要是在工业中，已经采用了从基因转录的RNA产物（cDNA）的大规模测序的策略。另一种有吸引力的方法是对基因进行特异性测序。人们可以利用玉米转座子激活子（Ac）插入低甲基化DNA（含有基因的基因组组分）中的倾向来鉴定基因作为Ac转座的位点（tac位点），然后对邻近转座子的DNA进行测序。这种方法的优点是，除了可以与现有数据库比较的序列之外，它还生成插入文库。在玉米基因组中的许多不同位置携带Ac的品系的收集将使研究人员能够筛选微妙的突变表型，特别是在获得关于基因在植物中表达的位置的信息之后。许多基因预期有轻微的影响，并可能错过了从传统的转座子诱变筛选设计，以确定总的变化在phenotype.Taking强大的胚乳遗传学的玉米，一个简单而有效的Ac转座试验的基础上，充分研究胚乳标记bz（青铜色）和wx（蜡质）已经开发。已经产生了超过1200个独立的Ac转座子的集合，并且这些Ac位点中超过三分之一已经相对于供体基因座进行了定位。与此同时，最初用于人类基因组的柄状PCR方法已适用于分离插入（tac位点）附近的DNA。通过对tac位点进行测序，例如在编码硫转运蛋白、MAPKK和倍半萜合酶的基因中已经鉴定出插入物。然而，因为Ac具有转座到紧密连锁位点的强烈倾向，所以大约一半的tac位点与9号染色体上的wx连锁。显然，希望从基因组中的不同起始位点动员Ac。为此，正在开发易于转化和再生的合适玉米品系。这些系将用携带经修饰以促进tac DNA分离的Ac元件的构建体转化。该构建体应整合在基因组中的随机位点，为将来的Ac动员提供起始平台。(b)基于tac位点的序列、Ac插入突变的表型和基因表达模式，阐明了一组50个基因的功能。(c)对应于玉米基因组中独特序列的那些基因的图谱位置。(d)转基因玉米品系的开发，将有助于将来从基因组中的任何位置分离基因（tac位点）。