EDGE FGT: Development of functional genomics tools in cactophilic Drosophila

EDGE FGT：嗜果蝇功能基因组学工具的开发

• 批准号: 2220279
• 负责人: Luciano Matzkin
• 金额: $ 115.54万
• 依托单位: University of Arizona
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-10-01 至 2026-09-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2220279&HistoricalAwards=false
• 关键词: EDGE; FGT; Development; functional; genomics

The genome of organisms can have thousands to tens of thousands of genes coded in their DNA. For each gene, organisms can also produce alternative versions of a gene product (usually a protein) as well as being turned on in specific tissues or at specific times during development. Therefore, just possessing knowledge of the DNA sequence of an organism’s genome is not enough to understand the function of all its genes. To facilitate this, investigators over the past several decades have been able to manipulate or edit the genome of organisms using a variety of molecular tools. Unfortunately, these tools have traditionally only been available for a very selected group of laboratory model organisms. Although these model organisms have dramatically expanded our understanding of the function of genes during development, in specific tissues and sometimes all the way down to specific cell types, the limited number laboratory model species and generalist ecology has prevented the analysis of the function of genes in a comparative, evolutionary and ecological context. This project will leverage the extensive ecological knowledge of several cactus-breeding fly species in the genus Drosophila with their recently determined genome sequences to build a set of genome-manipulable strains. Using these modified strains of closely related species investigators will be able to assess the function of genes (e.g. turning them on/off in specific tissues/cells). Knowledge of this new toolkit and its application will be disseminated via publications, a public website with detailed protocols and videos as well as an annual workshop.The central aim of this project is to enhance the power of investigators to assess the relationship between genotypes and phenotypes in an ecological and comparative context by creating a customizable transgenic system. Here investigator Matzkin proposes to build a transgenic toolkit for the existing diverse community utilizing the ecological model system of cactophilic Drosophila. This system offers a unique opportunity to integrate genomics, functional genomics, transcriptomics and phenomics in an ecological and comparative context. Cactophilic Drosophila are endemic to the Americas and oviposit, develop, and feed as adults in the necrotic tissues of a variety of cactus species. The transgenic toolkit will be built using the three most widely used cactophiles, D. mojavensis and D. arizonae from North America and D. buzzatii from South America; using the recently generated de novo chromosome level genome assemblies and annotations of each of the four D. mojavensis cactus host populations, of two ecologically divergent D. arizonae populations and a new comprehensive annotation of D. buzzatii. To build the toolkit PI Matzkin will: 1) generate lines with attP integration sites in several chromosomes using CRISPR; 2) use these lines to incorporate C31 integrase in the X chromosome of all three species; 3) perform detailed tissue, developmental and environment-dependent transcriptional analysis to identify driver loci; 4) generate tissue specific GAL4 driver lines and a UAS-GFP reporter; 5) generate a UAS-RNAi plasmid construct; and 6) disseminate the toolkit using the National Drosophila Species Stock Center, a dedicated online repository for information (cactusflybase) and in-person workshops.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

生物体的基因组可以在其DNA中具有数千至成千上万的基因。对于每个基因，生物还可以生成基因产物的替代版本（通常是蛋白质），并在发育过程中在特定时期内打开。因此，仅拥有有机体基因组的DNA序列的知识不足以理解其所有基因的功能。为了促进这一点，在过去的几十年中，研究人员能够使用各种分子工具来操纵或编辑生物体的基因组。不幸的是，这些工具传统上仅适用于非常精选的一组实验室模型生物。尽管这些模型生物已经大大扩展了我们对发展过程中基因功能，在特定组织中，有时一直延续到特定细胞类型的理解，但有限的实验室模型物种和通用生态学已经阻止了比较，进化和生态环境中基因功能的分析。该项目将利用果蝇属中几种仙人掌繁殖的蝇物种的广泛生态知识，并以其最近确定的基因组序列来构建一组基因组可操作菌株。使用这些修饰的密切相关物种研究者的菌株将能够评估基因的功能（例如，在特定的组织/细胞中打开/关闭它们）。对该新工具包及其应用的了解将通过出版物进行传播，该公共网站具有详细的协议和视频以及年度研讨会。该项目的主要目的是通过创建可定制的转基因系统来增强研究人员在生态和比较上下文中评估基因型和表型之间的关系。在这里，使用仙人掌果蝇的生态模型系统为现有潜水员社区建立转基因工具包的研究者提案。该系统提供了一个独特的机会，可以在生态和比较背景下整合基因组学，功能基因组学，转录组学和现象学。仙人掌果蝇对美洲和卵果是成年人在多种仙人掌种的坏死组织中成年后发展和喂养。转基因工具包将使用来自北美的三种最广泛使用的D. Mojavensis和D. Arizonae以及来自南美洲的D. Buzzatii建造；使用最近生成的从头染色体水平的基因组组件和四个D. mojavensis仙人掌宿主种群的注释，这是两个生态不同的Arizonae种群的，以及对Buzzatii的新综合注释。为了构建工具包Pi Matzkin将：1）使用CRISPR在几个染色体中与ATTP集成位点生成线路；使用这些线将整合在所有三个物种的X染色体中的C31组合在一起； 3）执行详细的组织，发育和环境依赖性转录分析以识别驱动器位置； 4）生成组织特定的GAL4驱动线和UAS-GFP报告基因； 5）产生UAS-RNAi质粒构建体；和6）使用国家果蝇物种储备中心，专门的在线存储库（仙人掌flybase）和面对面的研讨会来传播工具包。该奖项反映了NSF的法定任务，并通过使用基金会的知识分子和更广泛的影响审查审查标准来通过评估来诚实地诚实地支持。