EAGER: Engineering Chromosomal Deletions in Arabidopsis Plants using Unequal Crossovers

EAGER：使用不等交叉在拟南芥植物中进行染色体缺失工程

• 批准号: 1407063
• 负责人: Patrick Krysan
• 金额: $ 15万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1407063&HistoricalAwards=false
• 关键词: EAGER; Engineering; Chromosomal; Deletions; Arabidopsis

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.

人类依赖植物作为食物、燃料和纤维的重要来源。 因此，了解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，植物科学家的工具箱中缺少的是一种实用的策略，用于在染色体上DNA的选定位置上精确删除基因。 探索性研究（EAGER）项目的早期概念拨款将通过测试一种有前途的新策略来解决这一缺陷，该策略用于在植物中产生靶向基因组缺失。 由此产生的技术有可能被广泛采用，作为探索植物基因功能的标准工具。 此外，该项目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种独特的拟南芥突变体的分离，该突变体携带IV号染色体上三个串联重复基因的精确缺失。 这种缺失似乎是由位于基因组中相距25个碱基的T-DNA插入所指导的不相等减数分裂交换引起的。 通过PCR筛选2，000株F2植物以寻找重组子，发现了这种缺失。由于在先前发表的工作中只观察到一个这种缺失的实例，因此不可能准确估计这些缺失发生的速率。 为了确定该系统用于进行基因组工程的潜在价值，将进行以下研究：1. 测量T-DNA定向缺失在整个基因组中的几个位点发生的速率。 确定T-DNA结构对缺失频率的影响。 产生先前表征的缺失的T-DNA插入具有反向重复结构。 研究人员将探索发夹形成能力和缺失率之间的相关性。如果成功，这项研究最终可能导致基因组工程方法的发展，该方法可应用于包括作物在内的广泛植物物种。