Analysis of Genetic and Environmental Inputs into Accurate and Inaccurate Double Strand Break Repair in Plants

PI：J.L.Bennetzen(佐治亚大学)含有工程染色体断裂结构的植物将被用作研究系统，以研究双链DNA断裂(DSB)不准确修复的性质，以及影响不准确修复性质的遗传或环境因素。DSB修复，在从细菌到动植物的所有有机体中，都是高度准确的。这种精确度对于复制染色体并传递给后代来说是必要的，因为这些断裂是如此频繁，如果不修复就是致命的。然而，DSB的不准确修复确实会发生，导致断裂部位的微小缺失、插入或其他结果。以前的一些研究表明，不准确的DSB修复事件的相对大小和频率可能因物种而异，这种差异可能是基因组大小变化速率和序列变化速率差异的原因。这个项目将使用一个受调控的DSB系统，涉及从植物中表达的I-SCEI酶，然后将这些植物与具有I-SCEI切割位置的其他植物杂交。在初步实验中，这个杂交的后代已经被证明在I-SCEI位置经历DSB，有时在该位置有不准确的修复。研究人员将对这些后代植物中的许多I-SCEI位点进行DNA测序，以查看在切割位点发现了哪些缺失、插入等。这项工作是在四个草种(玉米、珍珠谷子、水稻和高粱)上进行的，它们的基因组大小和基因组稳定性非常不同，以确定这是否会影响不准确的DSB修复的性质或频率。I-SCEI位点也整合在不同植物的不同染色体位置上，因此人们可以研究染色体位置对修复结果的影响。最后，研究人员将在发育的不同时期，在不同的环境压力(例如，高紫外线强度或高臭氧水平)下，以及在已知DNA修复过程或染色质结构突变的遗传背景下，确定不准确的DSB修复的性质。综上所述，这些研究将提供对植物基因组如何进化的见解，特别是DSB修复如何参与这一过程，以产生自然选择所作用的自然多样性。这项研究将为理解基因组如何变化提供一种强大的方法和许多新颖的见解。这将有助于更好地理解生物学家如何解释和影响遗传多样性。该项目的许多直接参与者，特别是博士后、研究生和本科生，将接受多学科方法的直接培训，以解决现代遗传学中的一个核心问题：基因组如何变化，以及这种变化对基因组中基因的当前和未来功能有什么影响。该项目和成果的描述将在为该项目创建的网站上提供，其呈现风格使从K-12学生到普通公众再到活跃的研究科学家的所有有关各方都能理解这些信息。原始数据和最先进的结果也将在公众可访问的数据库中提供(例如，用于序列信息的GenBank和用于详细分析的同行评审出版物)，包括项目网站。生物材料(转基因植物的种子、I-SCEI方法的构建等)将从PI的实验室获得，直到可以确定一些更永久的维护资源。了解DNA变异，特别是机制和比率/结果，对于任何对遗传学或基因组的成熟理解至关重要，动物、人类、植物、真菌、病毒和微生物研究人员都有同样的看法，因此预计该项目的培训、推广和数据丰富部分将造福最广泛的科学界和普通公众。

PI: J. L. Bennetzen (University of Georgia)Plants containing engineered chromosome breakage constructs will be used as a study system to investigate the nature of inaccurate repair of double strand DNA breaks (DSB), and the genetic or environmental factors that affect the nature of inaccurate repair. DSB repair, in all organisms from bacteria to plants and animals, is highly accurate. This accuracy is necessary to allow chromosomes to be replicated and passed on to subsequent generations of progeny because these breaks are so frequent and are lethal if unrepaired. However, inaccurate repair of DSB does occur, leading to small deletions, insertions or other outcomes at the break site. Some previous studies have suggested that the relative sizes and frequencies of the inaccurate DSB repair events may vary from species to species, and that this variance might be responsible for differences in the rate at which genomes change size and for differences in their rate of sequence change. This project will use a regulated DSB system, involving the enzyme I-SceI expressed off a transgenic construct in plants that are then crossed to other plants that have a cutting site for I-SceI. The progeny of this cross have been shown, in preliminary experiments, to undergo DSB at the I-SceI site, and to sometimes have inaccurate repair at that site. The I-SceI site in many of these progeny plants will be DNA sequenced by the investigators to see what kind of deletions, insertions, et cetera, are found at the cleavage site. This work is being done in four grass species (maize, pearl millet, rice, and sorghum), with very different genomic sizes and genomic stability, to see if this influences the nature or frequency of inaccurate DSB repair. The I-SceI sites are also integrated at various different chromosomal locations in different plants, so one can investigate chromosome position effects on repair outcomes. Finally, the investigators will determine the nature of inaccurate DSB repair at different times in development, under different environmental stresses (e.g., high UV light intensity or high ozone levels) and in genetic backgrounds that have known mutations in DNA repair processes or chromatin structure. Taken together, these studies will provide insights into how plant genomes evolve, and particularly how DSB repair participates in this process, to generate the natural diversity on which natural selection acts. This research will provide a powerful approach, and numerous novel insights, for understanding how genomes change. This will lead to a better understanding of how genetic diversity can be interpreted and influenced by biologists. The many direct participants in the project, especially the postdoctoral fellow, graduate student and undergraduate students, will receive direct training in a multi-disciplinary approach to a core question in modern genetics: how do genomes change, and what effect does that change have on current and future function of genes in that genome. The description of the project and of the results will be available at a web site that will be created for this project, with a presentation style that makes the information digestible for all levels of interested parties, from K-12 students, to the general public, to active research scientists. Raw data and the most advanced results will also be provided in public-accessible databases (e.g., GenBank for sequence information and peer-reviewed publications for detailed analysis), including the project web site. Biological materials (seeds of transgenic plants, constructs for the I-SceI approach, etc.) will be available from the PI's lab until the time that some more permanent maintenance resource can be identified. Understanding DNA variation, particularly the mechanisms and rates/outcomes, is vital for any mature understanding of genetics or genomes, and is shared by animal, human, plant, fungal, viral and microbial researchers, so it is expected that the training, outreach and data enrichment components of this project will benefit the broadest scientific community and general public.