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

水的可获得性对动物的活动和分布模式有显著的影响。蒸发失水导致的干燥是最常见的脱水机制，也是陆地动物面临的普遍问题。大多数动物物种不耐受严重的水分胁迫。这项研究将在从分子到生物的水平上解决在水分胁迫期间保护动物的精确机制。了解这些保护机制可能会对广泛的科学界产生转译应用。例如，研究重要遗传模型的科学家能够通过强制干燥来延长幼年动物阶段的储存时间，这将对更经济地维持这些动物具有实际好处。这一优势将延伸到国家库存中心和教学实验室。对脱水敏感的生物体进行工程脱水耐受性是复杂的，但可以为在实验室培养中保持昂贵的有价值的活生物体提供干燥存储的可能性。此外，一些发现的生物医学意义可能是重大的。干燥细胞的储存将增加替代细胞在许多临床应用和再生医学中的可用性。在教育方面，该项目将促进对不同背景的研究生和本科生的培训。作为该项目的一部分，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、国家实验室科学家和希望获得新技能的学术人员)进行通识教育。这项合作研究将评估来自卤虫脱水生物胚胎的多个晚期胚胎发生丰富(LEA)蛋白对脱水耐受性的影响。该项目将完善和扩大对LEA蛋白质如何促进目标生物结构在从充分水化到水含量为2%或更少的连续体的各种水化状态下的稳定的理解。假说将用分离的大分子、脂质体、昆虫细胞系和黑腹果蝇幼虫进行验证。一种新的假设是，单个LEA蛋白所显示的功能是多方面的，这取决于干燥的严重程度。单个LEA蛋白的这种功能可塑性将代表着随着水合状态的变化而保护生物结构的新范例。一种新的植物LEA蛋白(AfrLEA6)已被克隆、测序和表达，该蛋白与最近发现的一种植物LEA蛋白具有较高的序列同源性，该蛋白与一种新的植物LEA蛋白有关，该蛋白对干燥损伤具有长期保护作用。AfrLEA6将稳定地导入昆虫细胞系，以探索在存在和不存在其他共转染的LEA蛋白和稳定糖的情况下，该蛋白是否会延长长期的脱水耐受性。最后，将创造出能够转基因表达多种LEA蛋白的果蝇品系。由于黑腹蠕虫幼虫阶段自然含有高水平的内源性海藻糖(一种已知与LEA蛋白质协同提供保护的糖)，幼虫将被用于测试该物种对干燥的耐受性改善，这种物种通常对干燥敏感，并且其基因组中不包含LEA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.