EAGER: Engineering Chromosomal Deletions in Arabidopsis Plants using Unequal Crossovers

人类依赖植物作为食物、燃料和纤维的重要来源。因此，了解支配植物生长和发育的生物学原理具有很大的实用价值。植物遗传学为理解植物生物学提供了最有效的方法之一。基因组科学的最新进展提供了一套丰富的工具和资源，大大加快了植物遗传学研究的步伐。然而，这位植物学家的工具箱中缺少的是一种实用的策略，可以通过工程技术精确删除染色体上DNA中选定位置的基因。这一早期概念的探索性研究拨款(AGERGE)项目将通过测试一种有前景的新策略来解决这一不足，以产生植物中的定向基因组删除。由此产生的技术有可能被广泛采用，成为探索植物基因功能的标准工具。此外，该项目还将为研究生和本科生提供培训机会。该研究小组最近描述了一个独特的拟南芥突变体的分离，该突变体携带着四号染色体上三个重复的基因的精确缺失。这种缺失似乎是由基因组中相距25kb的T-DNA插入导致的不相等的减数分裂交叉引起的。通过聚合酶链式反应筛选2,000株F2代植株寻找重组子，发现了该缺失。因为在之前发表的工作中只观察到了这种缺失的一个实例，所以不可能准确地估计这些缺失发生的速度。为了确定该系统在进行基因组工程方面的潜在价值，将进行以下研究：1.测量T-DNA在整个基因组中多个基因座发生定向缺失的比率。确定T-DNA结构对缺失频率的影响。产生先前特征缺失的T-DNA插入片段具有反向重复结构。发夹形成能力和缺失率之间的相关性将被探索。如果这项研究成功，这项研究最终可能导致基因组工程方法的发展，这些方法可以应用于包括农作物在内的广泛植物物种。

Humans are dependent on plants as critical sources of food, fuel, and fiber. Understanding the biological principles that govern the growth and development of plants therefore has great practical value. The science of plant genetics provides one of the most productive approaches to understanding plant biology. Recent advances in genome science have provided a rich suite of tools and resources that have greatly accelerated the pace of plant genetics research. Missing from the plant scientist's toolbox, however, is a practical strategy for engineering precise deletions of genes at chosen positions in the DNA on a chromosome. This Early-concept grant for Exploratory Research (EAGER) project will address this deficiency by testing a promising new strategy for producing targeted genome deletions in plants. The resulting technology has the potential to be widely adopted as a standard tool for exploring gene function in plants. In addition, the project will provide training opportunities for students at the graduate and undergraduate levels. The research team recently described the isolation of a unique Arabidopsis thaliana mutant carrying the precise deletion of three tandemly-duplicated genes on chromosome IV. This deletion appears to have been caused by an unequal meiotic crossover directed by T-DNA insertions located 25 kilobases apart in the genome. This deletion was found by screening 2,000 F2 plants by PCR to search for recombinants. Because only one instance of this deletion was observed in the previously published work, it is impossible to accurately estimate the rate with which these deletions occur. In order to determine the potential value of this system for performing genome engineering the following studies will be performed:1. Measure the rate with which T-DNA directed deletion occurs at several loci throughout the genome.2. Determine the effect that T-DNA structure has on deletion frequency. The T-DNA insertions that produced the previously characterized deletion had an inverted-repeat structure. The correlation between hairpin-forming ability and deletion rate will be explored.If successful, this research could ultimately lead to the development of genome engineering methods that could be applied to a wide range of plants species, including crop plants.