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成效的方法之一。基因组科学的最新进展提供了丰富的工具和资源，大大加快了植物遗传学研究的步伐。然而，在植物科学家的工具箱中缺少的是一种实用的策略，即在染色体上DNA的选定位置精确地删除基因。探索性研究（EAGER）项目的早期概念资助将通过测试一种在植物中产生靶向基因组缺失的有前途的新策略来解决这一缺陷。由此产生的技术有可能作为探索植物基因功能的标准工具被广泛采用。此外，该项目将为研究生和本科生提供培训机会。研究小组最近描述了一种独特的拟南芥突变体的分离，该突变体携带4号染色体上三个串联复制基因的精确缺失。这种缺失似乎是由位于基因组中相隔25千碱基的T-DNA插入所导致的不平等减数分裂交叉引起的。对2000株F2植株进行PCR筛选，发现了这种缺失。因为在先前发表的工作中只观察到这种缺失的一个实例，因此不可能准确估计这些缺失发生的速率。为了确定该系统在基因组工程中的潜在价值，将进行以下研究：1。测量T-DNA定向缺失在整个基因组中几个位点发生的速率。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插入具有倒置重复结构。探讨发夹形成能力与缺失率之间的关系。如果成功的话，这项研究可能最终导致基因组工程方法的发展，这种方法可以应用于包括农作物在内的各种植物物种。

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.