Arabidopsis 2010: A Transposon-Based System for Site-Specific Recombination in Arabidopsis

本项目的目标是开发一种在拟南芥中进行高效的同源依赖的位点特异性重组(SSR)的方法。所使用的方法是基于转座元件的重组诱导特性，以及将重组底物输送到植物细胞的替代方法。在先前的研究中，pI和co-pI已经表明DNA和RNA转座元件可以刺激重组。首先，玉米Ac/ds转座子的切除极大地诱导了植物中的同源重组，包括拟南芥。其次，反转录转座子可以产生高水平的cdna，很容易与基因组序列重组。该项目将开发包含部分重叠的、可见的和可选择的标记基因片段的双组分(受体和供体)转基因构建体。此外，受体组分还包含插入在标记基因之间的玉米DS元件。受体结构被整合到拟南芥基因组中；当Ac/DS转座酶表达时，DS切除在受体结构中产生重组热点。供体构建体序列通过几种可供选择的方法进入植物细胞，包括农杆菌T-DNA转化、基因轰击、异位染色体定位和反转录转座子产生的cDNA。受体和供体序列的重组可以通过可见标记在体细胞中检测到，并可以对传递给后代的事件进行遗传选择。可遗传重组事件将通过分子和遗传分析来表征，以更好地了解植物细胞中的SSR机制。项目结果将在以下网站上查阅：http://www.public.iastate.edu/~voytas/rec_arab.html.所有试剂(转化品系的种子、质粒载体)都将根据要求提供。同源性依赖的重组是许多生物遗传修饰的有力工具；然而，常规SSR方法尚不适用于高等植物。拟南芥2010项目的一个明确目标是开发定向突变和SSR的方法。该项目的成功完成将为基础研究和实际应用提供一种对植物基因组进行精确改变的重要新手段。

The goal of this project is to develop a method for efficient homology-dependent site-specific recombination (SSR) in Arabidopsis. The approach used is based on the recombination-inducing properties of transposable elements, coupled with alternative methods of delivering recombination substrates to plant cells. In previous research, the PI and co-PI have shown that DNA and RNA transposable elements can stimulate recombination. First, excision of maize Ac/Ds DNA transposons greatly induces homologous recombination in plants, including Arabidopsis. Second, retrotransposons can generate high levels of cDNA that recombine readily with genomic sequences. This project will develop two-component (recipient and donor) transgene constructs that contain partially-overlapping visible and selectable marker gene fragments. The recipient component contains, in addition, a maize Ds element inserted between the marker genes. The recipient construct is integrated into the Arabidopsis genome; upon expression of Ac/Ds transposase, Ds excision generates a recombination hotspot in the recipient construct. The donor construct sequences are delivered into the plant cells via several alternative methods, including Agrobacterium T-DNA transformation, particle bombardment, ectopic chromosomal position, and retrotransposon-generated cDNA. Recombination of recipient and donor sequences can be detected in somatic cells by the visible marker, and events transmitted to progeny can be genetically selected. Heritable recombination events will be characterized by molecular and genetic analysis to gain a greater understanding of SSR mechanisms in plant cells. Project results will be available at the following web site: http://www.public.iastate.edu/~voytas/rec_arab.html. All reagents (seeds of transformed lines, plasmid vectors) will be made available upon request.Homology-dependent recombination is a powerful tool for genetic modification in many organisms; however, methods for routine SSR are not yet available for higher plants. An explicit goal of the Arabidopsis 2010 project is the development of methods for directed mutations and SSR. Successful completion of this project will provide an important new means for making precise changes in plant genomes for both fundamental research and practical applications.