Nested Deletions: A New Tool for Plant Genomics Research

植物基因组中存在大量的局部序列重复和同源基因簇。为了简化对这些重复和基因簇的分析，该项目将开发一种新的遗传技术，以快速有效地产生染色体缺失。这种方法是基于这样的发现，即转座因子可以参与替代转座途径，产生新的重组产物，包括大的缺失和重复。本文所述的系统利用转基因构建体，所述转基因构建体含有在I/dSpm转座子内串联取向的玉米Ac/Ds转座子末端（Ned 1;巢式缺失1）。将Ned 1构建体转化到玉米中;随后的杂交将引入En/Spm转座酶以将Ned 1移动到各个基因组位置，并引入Ac转座酶以激活缺失过程。Ac转座酶作用于Ned 1内的Ac末端，产生一组无限的嵌套缺失，其中一端锚定在转基因位点。Ned 1构建体包含用于检测Ned 1转座和Ac-诱导缺失的标记基因，以及用于通过质粒拯救容易克隆缺失终点的序列。该项目的预期成果是生产含有Ned 1转基因构建体的玉米植株。将测试这些植物及其后代的1）通过En/Spm转座酶的Ned 1转座; 2）通过Ac转座酶的缺失诱导; 3）缺失向后代植物的传递。此外，Ned 1转基因系、含En/Spm和Ac的系将与常用的玉米自交系[B73]回交，以使这些材料对研究界最有用。这种方法可以扩展到生产一组在整个基因组的分散位点含有Ned 1元件的玉米品系，其可以用于分离基因组的特定区域中的缺失和其他重排。该项目将产生含有Ned 1转基因构建体的玉米植株。2.将测试Ned 1转基因植物及其后代的a）由En/Spm转座酶诱导的Ned 1转座; B）由Ac转座酶诱导的缺失的形成; c）缺失向后代植物的传递。3. Ned 1转基因系、含En/Spm和Ac的系将与常用的玉米自交系回交[B73]，以使这些材料对研究界最有用。联系人信息：托马斯彼得森，2206分子生物学，爱荷华州州立大学，艾姆斯，IA 50011thomasp@iastate.edu

Plant genomes are laden with local sequence duplications and clusters of homologous genes. To simplify the analysis of these duplications and gene clusters, this project will develop a new genetic technology to generate chromosomal deletions quickly and efficiently. This approach is based on the finding that transposable elements can participate in alternative transposition pathways that generate novel recombination products, including large deletions and duplications. The system described here utilizes a transgene construct containing maize Ac/Ds transposon ends in tandem orientation within a I/dSpm transposon (Ned1; Nested deletions 1). The Ned1 construct will be transformed into maize; subsequent crosses will introduce the En/Spm transposase to mobilize Ned1 to various genomic locations, and the Ac transposase to activate the deletion process. The action of Ac transposase on the Ac termini within Ned1 generates an unlimited set of nested deletions with one end anchored at the transgene locus. The Ned1 construct contains marker genes for detection of both Ned1 transpositions and Ac--induced deletions, as well as sequences for easy cloning of deletion endpoints via plasmid rescue. The expected outcome of this project is the production of maize plants containing the Ned1 transgene construct. These plants and their progeny will be tested for 1) transposition of Ned1 by En/Spm transposase; 2) induction of deletions by Ac transposase; 3) transmission of deletions to progeny plants. Additionally, the Ned1 transgenic lines, En/Spm- and Ac-containing lines will be backcrossed to a commonly-used maize inbred line [B73] in order to make these materials most useful for the research community. This approach could be extended to the production of a set of maize lines containing Ned1 elements at dispersed sites throughout the genome that could be used to isolate deletions and other rearrangements in specific regions of the genome.Deliverables: 1. This project will generate maize plants containing the Ned1 transgene construct. 2. Ned1 transgenic plants and their progeny will be tested for a) transposition of Ned1 induced by En/Spm transposase; b) formation of deletions induced by Ac transposase; c) transmission of deletions to progeny plants. 3. Ned1 transgenic lines, En/Spm- and Ac-containing lines will be backcrossed to a commonly-used maize inbred line [B73] in order to make these materials most useful to the research community. Contact Information for Deliverables: Thomas Peterson, 2206 Molecular Biology, Iowa State University, Ames, IA 50011thomasp@iastate.edu