Exon-Trap Sequencing Using Transposons in Arabidopsis

摘要利用转座因子Ac和Ds，建立了拟南芥体内基因捕获技术。我们改进的Ds元件（DsG）包含一个β -葡萄糖醛酸酶报告基因，该基因具有剪接受体信号，允许多帧基因融合（“外显子陷阱”）。当这个Ds元素插入染色体基因时，染色体基因的表达模式被β -葡糖醛酸酶报告基因模仿。由于表达依赖于基因融合，导致报告基因表达的插入也会导致基因破坏。利用一种新的选择方案，将产生5000株植物，每株植物在基因组的不同位置携带一个插入。插入基因将通过染色每株植物的后代来鉴定报告基因表达：15-20%的DsG插入导致报告基因表达，因此建议的收集应包括插入500- 1,000个基因。我们将利用这些收集来开发一种外显子陷阱测序技术，以实现每个外显子陷阱标签识别的染色体基因部分的快速测序。每个插入对应的染色体外显子将通过RACE PCR扩增，并确定其序列。尽可能多的外显子将通过锚定的YAC contigs和重组近交系来定位。将建立一个DsG插入系数据库，每个插入系都具有表达模式、突变表型和图谱位置以及部分外显子序列的特征。该数据库可以与现有的基因组和cDNA序列数据库集成。***拟南芥基因组鉴定的一个主要目标是通过cDNA和基因组测序、基因寻找算法和图谱定位来确定基因的功能。同源性搜索有望实现这一目标，寻找那些与其他生物序列相似的基因。然而，在这些测序基因的大集合中，大多数基因产物将保持匿名。在拟议的研究中，通过确定它们的表达模式，并通过观察基因破坏导致的突变表型，将功能分配给许多这些基因。这一建议将成为建立一个大型插入线数据库的基础，因为其他实验室使用“起始”线来生成自己的收集。这将允许将功能分配给许多仅通过DNA序列识别的基因，并提供广泛使用的遗传资源。% % %

Abstract 9408042 An in vivo gene-trapping technique has been developed in Arabidopsis using the transposable elements Ac and Ds. Our modified Ds element (DsG) contains a Beta-glucuronidase reporter gene that is equipped with splice acceptor signals to allow multi- frame gene fusions (an "exon-trap"). When this Ds element inserts into a chromosomal gene, the pattern of chromosomal gene expression is mimiced by the Beta-glucuronidase reporter gene. Because expression depends on gene fusion, insertions that result in reporter gene expression will also result in gene disruption. Using a novel selection scheme, a collection of 5,000 plants will be generated that will each carry an insertion at a different location in the genome. Insertions into genes will be identified by staining the progeny of each plant for reporter gene expression: 15-20% of DsG insertions result in reporter gene expression, so that the proposed collection should include insertions into 500- 1,000 genes. We will use this collection to develop an exon-trap sequencing technique to enable the rapid sequencing of a portion of the chromosomal gene identified by each exon-trap tag. The chromosomal exon corresponding to each insertion will be amplified by RACE PCR, and its sequence determined. As many of the exons as possible will be mapped via anchored YAC contigs and recombinant inbred lines. A database of DsG insertion lines will be developed, each characterized by expression pattern, mutant phenotype and map location, as well as partial exon sequence. This database can be integrated with existing genomic and cDNA sequence databases. *** A major goal in the characterization of the Arabidopsis genome is to assign a function to genes identified by cDNA and genomic sequencing, gene-finding algorithms, and map location. Homology searches are expected to fulfill this goal for those genes that have similarity to sequences from other organisms. However, a majority of gene products will remain an onymous in these large collections of sequenced genes. In the proposed study, a function will be assigned to many of these genes by determining their patterns of expression, and by observing the mutant phenotype that results from gene disruption. This proposal will form the basis for establishing a large database of insertion lines, as other laboratories use the "starter" lines to generate collections of their own. This will allow a function to be assigned to many of the genes identified by DNA sequence alone, as well as providing a genetic resource of widespread use. %%%