EDGE FGT: Development of functional genomics tools in cactophilic Drosophila

生物体的基因组在其DNA中可以有数千到数万个基因编码。对于每个基因，生物体还可以产生基因产物的替代版本(通常是蛋白质)，并在特定组织中或在发育过程中的特定时间被激活。因此，仅仅拥有生物体基因组的DNA序列知识并不足以了解其所有基因的功能。为了促进这一点，在过去的几十年里，研究人员已经能够使用各种分子工具来操纵或编辑有机体的基因组。不幸的是，这些工具传统上只适用于非常精选的一组实验室模式生物。尽管这些模式生物极大地扩展了我们对基因在发育过程中的功能的理解，在特定的组织中，有时甚至一直延伸到特定的细胞类型，但有限的实验室模式物种和通才生态学阻碍了在比较、进化和生态背景下分析基因的功能。该项目将利用果蝇属几个仙人掌繁殖苍蝇物种的广泛生态学知识及其最近确定的基因组序列来构建一组可进行基因组操作的菌株。使用这些密切相关物种的改良菌株，研究人员将能够评估基因的功能(例如，在特定组织/细胞中打开/关闭它们)。这一新工具包及其应用的知识将通过出版物、具有详细方案和视频的公共网站以及年度工作坊传播。该项目的中心目标是通过创建一个可定制的转基因系统，增强研究人员在生态和比较背景下评估基因和表型之间关系的能力。在这里，研究人员Matzkin建议利用嗜食果蝇的生态模型系统为现有的多样化群落建立一个转基因工具包。这个系统提供了一个独特的机会，在生态和比较的背景下整合基因组学、功能基因组学、转录组学和表观组学。喜食仙人掌的果蝇是美洲特有的物种，成虫在各种仙人掌种类的坏死组织中产卵、发育和取食。转基因工具包将使用三种使用最广泛的仙人掌，北美的莫杰文氏丝虫和阿利佐尼丝虫和南美洲的布氏丝虫；使用最近产生的从头开始染色体水平的基因组组合和四个莫杰文氏丝虫仙人掌寄主种群的每一个的注释，两个生态上不同的阿利佐尼丝虫种群和一个新的全面的布氏丝虫种群的注释。为了建立工具包，Pi Matzkin将：1)使用CRISPR产生在几条染色体上具有attP整合位点的系；2)利用这些系将C31整合酶整合到所有三个物种的X染色体上；3)进行详细的组织、发育和环境相关的转录分析以确定驱动基因座；4)产生组织特异性的GAL4驱动系和UAS-GFP报告；5)产生UAS-RNAi质粒构建体；和6)使用国家果蝇物种储备中心传播工具包，这是一个专门的在线信息库(仙人掌飞库)和面对面的工作室。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.