Nested Deletions: A New Tool for Plant Genomics Research

植物基因组中含有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基因技术，以快速有效地产生染色体缺失。这一方法是基于这样的发现，即转座元件可以参与产生新的重组产物的替代转座途径，包括大的缺失和复制。该系统利用含有玉米Ac/DS转座子末端的转基因构建物，在i/dSpm转座子内串联定位(Ned1；嵌套缺失1)。Ned1构建体将被转化到玉米中；随后的杂交将引入EN/SPM转座酶来将Ned1动员到不同的基因组位置，并引入ac转座酶来激活缺失过程。在NED1中，Ac转座酶对Ac末端的作用产生了一组无限的嵌套缺失，其中一端固定在转基因位点上。Ned1结构包含用于检测Ned1转座和Ac诱导的缺失的标记基因，以及用于通过质粒拯救轻松克隆缺失终点的序列。该项目的预期结果是生产出含有Ned1转基因构建物的玉米植株。这些植物及其后代将被检测：1)EN/SPM转座酶转座Ned1；2)AC转座酶诱导缺失；3)缺失传递给后代植株。此外，Ned1转基因系、En/Spm-系和Ac-系将回交到一个常用的玉米自交系[B73]，以便使这些材料对研究界最有用。这一方法可以扩展到生产一组在整个基因组分散位置含有Ned1元素的玉米品系，可以用来分离基因组特定区域的缺失和其他重排。交付：1.这个项目将产生含有Ned1转基因构建的玉米植株。2.Ned1转基因植株及其后代将被检测a)EN/SPM转座酶诱导的Ned1转座；b)ac转座酶诱导的缺失的形成；c)缺失的遗传给后代植株。3.将Ned1转基因系、含En/Spm和Ac的自交系回交到一个常用的玉米自交系[B73]，以使这些材料对研究界最有用。爱荷华州立大学2206分子生物学，Ames，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