Collaborative Research: Mechanisms of Tolerance to Severe Water Stress in Animals

水的供应对动物活动和分布模式有明显的影响。 由于蒸发失水导致的干燥是最常见的脱水机制，并且是陆生动物面临的普遍问题。 大多数动物不能忍受严重的水分胁迫。这项研究将解决从分子到生物体水平在水胁迫期间保护动物的精确机制。 了解这些保护机制可能对广泛的科学界具有转化应用。 例如，研究重要遗传模型的科学家能够通过实施干燥来延长幼仔阶段的储存时间，这对于更经济地维持这些动物具有实际好处。 这种优势将延伸到国家库存中心和教学实验室。 在脱水敏感的生物体中设计脱水耐受性是复杂的，但可以为在实验室培养中维持可能困难且昂贵的有价值的活生物体的干燥储存提供可能性。 此外，一些研究结果的生物医学影响可能是重要的。 干细胞的储存将增加许多临床应用和再生医学中替代细胞的可用性。在教育方面，该项目将促进来自各种背景的研究生和本科生的培训。 作为该项目一部分计划开展的外联活动将有助于对国际科学家（包括私营部门的生物技术研究人员、国家实验室的科学家和希望获得新技能的学术人员）进行一般教育。这项合作研究将评估多种晚期胚胎发育丰富（莱亚）蛋白质来源于卤虫franciscana的脱水胚胎对脱水耐受性的影响。 该项目将完善和扩展莱亚蛋白如何促进目标生物结构在不同水合状态下的稳定性的理解，沿着从完全水合到2%或更少的水含量的连续体。假设将用分离的大分子、脂质体、昆虫细胞系和果蝇幼虫进行测试。一个新的假设是，由个别莱亚蛋白质显示的功能是多方面的，这取决于脱水的严重程度。 单个莱亚蛋白的这种功能可塑性将代表随着水合状态变化而保护生物结构的新范例。 A.一个新的莱亚蛋白。franciscana（AfrLEA6）与最近发现的一种植物莱亚蛋白具有高度的序列同源性，该蛋白与抗干旱损伤的长期保护有关。 AfrLEA6将被稳定转染到昆虫细胞系中，以探索在存在和不存在其他共转染的莱亚蛋白和稳定糖的情况下，该蛋白是否将延长长期干燥耐受性。 最后，将建立转基因表达多种莱亚蛋白的果蝇品系。 由于D.黑腹果蝇天然含有高内源水平的海藻糖（已知与莱亚蛋白协同提供保护的糖），幼虫将用于测试该物种中改进的干燥耐受性，该物种通常是干燥敏感的并且在其基因组中不含莱亚基因。

Water availability has pronounced influences on animal activity and distribution patterns. Drying due to evaporative water loss is the most common mechanism for dehydration, and it is a universal problem confronting terrestrial animals. The majority of animal species do not tolerate severe water stress. This research will address precise mechanisms that protect animals during water stress at levels from the molecular to the organismal. Understanding these protective mechanisms may have translational application for broad scientific communities. For example, the ability for scientists working with important genetic models to prolong the storage of young animal stages by imposing desiccation would be of practical benefit for maintaining these animals more economically. The advantage would extend to national stock centers and teaching laboratories. Engineering desiccation tolerance in dehydration-sensitive organisms is complex, but could provide possibilities for dry storage of valuable live organisms that may be difficult and expensive to maintain in laboratory culture. Further, the biomedical implications of some findings could be significant. Storage of dried cells would increase the availability of replacement cells for many clinical applications and in regenerative medicine. Educationally, the project will facilitate the training of graduate students and undergraduates from a wide diversity of backgrounds. Outreach activities planned as part of the project will contribute to the general education of international scientists (including researchers in biotechnology from the private sector, National Laboratory scientists, and academic faculty wishing to acquire new skills). This collaborative research will evaluate the impact on desiccation tolerance of multiple Late Embryogenesis Abundant (LEA) proteins originating from anhydrobiotic embryos of the brine shrimp Artemia franciscana. The project will refine and expand the understanding of how LEA proteins promote stabilization of targeted biological structures at various hydration states along a continuum from full hydration to water contents of 2% or less. Hypotheses will be tested with isolated macromolecules, liposomes, insect cells lines, and larvae of Drosophila melanogaster. One novel hypothesis is that the function displayed by individual LEA proteins is multifaceted depending upon the severity of desiccation. Such functional plasticity for individual LEA proteins would represent a new paradigm for protection of biological structures as hydration state varies. A new LEA protein from A. franciscana (AfrLEA6), with high sequence homology to a plant LEA protein recently linked to long-term protection against desiccation damage, has been cloned, sequenced and expressed. AfrLEA6 will be stably transfected into insect cell lines to explore whether this protein will extend long-term desiccation tolerance, in the presence and absence of other co-transfected LEA proteins and stabilizing sugars. Finally, fly lines of Drosophila melanogaster will be created that transgenically express multiple LEA proteins. Because larval stages of D. melanogaster naturally contain high endogenous levels of trehalose (a sugar known to provide protection synergistically with LEA proteins), larvae will be used to test for improved tolerance to drying in this species that is generally desiccation-sensitive and does not contain LEA genes in its genome.