Genetic dissection and characterization of variation in copper resistance in Drosophila melanogaster

项目摘要 金属对人体健康有着复杂的影响。有些是正常发育所需的少量， 体内平衡和缺陷可能导致疾病。然而，对许多金属的过度暴露甚至会造成更大的 风险必需和非必需金属中毒可导致永久性神经系统疾病， 退化综合征和急性器官损伤的可能性。人类的大部分金属中毒 消费因工业废物处置不当而受到污染的食物和水， 充足的饮用水卫生设施。金属中毒也常常是产业工人的职业危害 但最常见的是，儿童和贫困社区的发病率最高， 金属中毒的长期后果。值得注意的是，遗传变异会影响对金属中毒的易感性- 然而，导致对金属中毒的抵抗力变化的基因组因素代表了一个关键因素。 研究领域。因为对金属中毒的抵抗力可能是一种基因复杂的特征， 可以通过对数量自然变异的全基因组解剖来获得。我们的主要目标是解剖 并使用黑果蝇（Drosophila melano）来表征抗铜中毒的遗传变异， gaster模型系统，它与人类共享几个保守的金属反应基因和途径。我们 将铜作为感兴趣的模型金属，因为其对正常细胞功能的关键要求和相似性 从铜代谢途径到必需金属（如锌）和非必需金属（如铅）的代谢途径。 通过Aim 1，我们将整合一个大规模的表型筛选，以了解组织铜抗性的变化， 特异性表达谱分析，以鉴定和表征有助于铜的基因座和调控变体 阻力与目标2，我们将研究在10个铜抗性的遗传结构的变化， 自然分离D.黑腹种群，并了解遗传背景和进化- 这些群体的遗传史影响铜相关等位基因变异的效应。目标3：我们 将使用基因组编辑和相互半合子分析来功能验证强候选基因 目的1和2中确定的，直接测试假设导致高或低的特定等位基因的影响， 在受控遗传背景下的铜抗性。这种综合方法利用（1）QTL定位，基因 表达分析，并在一个强大的参考作图群体中进行铜抗性的表达QTL定位， （2）自然分离群体铜抗性的群体分离分析; (3)通过靶向基因编辑验证和表征候选铜抗性基因。去- 总之，这些方法将提供对金属抗性遗传控制变异的详细了解 中毒这将最终提高我们对人类对金属中毒易感性的理解- 在治疗因环境污染而受到金属中毒伤害的人方面， 精神暴露或代谢疾病。

PROJECT SUMMARY Metals have complex effects on human health. Some are required in small amounts for normal development and homeostasis, and deficiencies can result in disease. However, overexposure to many metals poses even greater risks. Poisoning with essential and non-essential metals can lead to permanent neurological diseases, increased probability of degenerative syndromes, and acute organ injury. The bulk of metal poisoning in humans results from the consumption of food and water contaminated from inappropriate disposal of industrial wastes and in- adequate drinking water sanitation. Metal poisoning is also often an occupational hazard for industrial workers and miners, but most frequently, small children and poor communities suffer the highest incidence and most prolonged consequences of metal poisoning. Notably, genetic variation influences susceptibility to metal poison- ing, but the genomic factors that contribute to variation in resistance to metal poisoning represent a critically understudied area. Because resistance to metal poisoning is likely a genetically complex trait, substantial insight can be gained through genomewide dissection of quantitative natural variation. Our primary objective is to dissect and characterize the genetic variation underlying resistance to copper poisoning using the Drosophila melano- gaster model system, which shares several conserved metal-responsive genes and pathways with humans. We treat copper as a model metal of interest due to its critical requirement for normal cell function and the similarity of the copper metabolic pathway to that of both essential (such as zinc) and non-essential (such as lead) metals. With Aim 1 we will integrate a large-scale phenotyping screen for variation in copper resistance with tissue- specific expression profiling to identify and characterize loci and regulatory variants that contribute to copper resistance. With Aim 2 we will examine variation in the genetic architecture of copper resistance among 10 naturally segregating D. melanogaster populations, and understand how the genetic backgrounds and evolu- tionary history of these populations influences the effects of copper-associated allelic variation. With Aim 3, we will use genome editing and Reciprocal Hemizygosity Analysis to functionally validate strong candidate genes identified in Aims 1 and 2, directly testing the influence of specific alleles hypothesized to lead to high or low copper resistance in controlled genetic backgrounds. This integrated approach leverages (1) QTL mapping, gene expression analysis, and expression QTL mapping of copper resistance in a powerful reference mapping popu- lation, (2) bulked-segregant analysis of copper resistance in replicated naturally segregating populations, and (3) validation and characterization of candidate copper resistance genes through targeted gene editing. To- gether, these approaches will provide detailed insight into variation in the genetic control of resistance to metal poisoning. This will ultimately improve our understanding of susceptibility to metal poisoning in human popula- tions and foster innovation in the treatment of those who have been harmed by metal poisoning due to environ- mental exposure or metabolic disease.