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，燃料和纤维的关键来源。 因此，了解控制植物生长和发育的生物学原理具有巨大的实用价值。 植物遗传学科学提供了理解植物生物学的最有生产力的方法之一。 基因组科学的最新进展提供了一套丰富的工具和资源，这些工具和资源大大加快了植物遗传学研究的速度。 然而，植物科学家的工具箱缺失是一种实用策略，可以在染色体上在DNA中选择的基因进行精确缺失。 这项对探索性研究（急切）项目的早期概念赠款将通过测试一种有希望的新策略来解决植物中的靶向基因组缺失，以解决这一缺陷。 所得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级别的学生提供培训机会。 研究小组最近描述了一个独特的拟南芥突变体的隔离，该突变体携带了染色体IV上三个串联基因的精确缺失。 这种删除似乎是由基因组中25千碱基的T-DNA插入引导的不平等的减数分裂跨界造成的。 通过通过PCR筛选2,000个F2植物来寻找重组剂，发现了这种缺失。因为在先前发表的工作中只观察到了这种删除的一个实例，因此无法准确估计这些删除发生的速率。 为了确定该系统对执行基因组工程的潜在值，将进行以下研究：1。 测量整个基因组中T-DNA定向缺失在几个基因座发生的速率2。 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插入具有倒重复结构。 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，这些方法可以应用于包括作物植物在内的各种植物物种。