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

大部分玉米基因组（90%）由重复DNA组成，其中很大一部分被甲基化。基因占基因组的比例不到3%，它们被发现在被称为低甲基化岛的低甲基化DNA的连续延伸中。由于玉米中的重复DNA含量，对整个玉米基因组（2.5xl09 bp）进行测序是不切实际的。一些实验室，主要是工业实验室，采用了对基因转录的RNA产物（cdna）进行大规模测序的策略。一个有吸引力的替代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（包含基因的基因组成分）的趋势来识别Ac转座的基因位点（tac位点），然后对转座子附近的DNA进行测序。这种方法的一个优点是，除了可以与现有数据库进行比较的序列外，它还生成一个插入库。在玉米基因组的许多不同位置收集携带Ac的品系将使研究人员能够筛选细微的突变表型，特别是在获得有关基因在植物中的表达位置的信息之后。许多基因预计有轻微的影响，并可能从传统的转座子突变筛选中遗漏，该筛选旨在确定表型的总体变化。利用玉米强大的胚乳遗传特性，基于已研究的胚乳标记bz（青铜）和wx（蜡质），建立了一种简单有效的Ac转位测定方法。已经产生了超过1200个独立的Ac转座，其中超过三分之一的Ac位点已经相对于供体位点进行了定位。与此同时，最初用于人类基因组的长柄PCR方法已被用于分离插入位点（tac位点）附近的DNA。通过对tac位点的测序，已经确定了插入，例如，在编码硫转运蛋白、MAPKK和倍半萜合成酶的基因中。这种Ac动员方案允许分离与供体位点有遗传关联或无遗传关联的tac位点。然而，由于Ac有很强的转位到紧密连接位点的倾向，大约一半的tac位点与9号染色体上的wx相连。显然，从基因组的不同起始位点调动Ac是可取的。为此，正在开发易于转化和再生的合适的玉米品系。这些细胞系将被转化为携带Ac元素的结构体，以促进tac DNA的分离。这种结构应该在基因组的随机位点整合，为未来的Ac动员提供起始平台。这一提议的预期结果是：(a)超过1000个玉米基因或基因片段的序列。(b)基于tac位点序列、Ac插入突变的表型和基因表达模式，对一组50个基因的功能进行了阐明。(c)与玉米基因组中独特序列相对应的基因的图谱位置。(d)开发转基因玉米品系，以便将来从基因组的任何位置分离基因（tac位点）。

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.