Studies of Antifreeze Proteins and Related Overwintering Adaptations in Arctic and Anarctic Insects

这是阿拉斯加大学费尔班克斯分校和普渡大学首席研究人员共同提出的建议。抗冻蛋白(AFP)存在于多种生物中，包括鱼、昆虫、蜘蛛、螨类、植物和细菌。虽然这些AFP之间存在巨大的结构差异，但它们似乎都通过类似的非缔合机制降低了水的冰点。然而，到目前为止，昆虫AFP是最活跃的，通常通过抑制冰核剂和跨角质层的无害冻结来防止易冻昆虫的冻结。然而，少数产生AFP的昆虫是耐寒的，AFP在这些昆虫中的作用尚不清楚。已鉴定了两种昆虫AFP。已从两种甲虫中描述了一种类型，但在种内和种间在序列等方面存在差异。直到最近的研究确定了18种具有AFP的物种，这些蛋白质在阿拉斯加或北极的昆虫中还没有报道。其中一些已被选择用于进一步研究，这为将对昆虫afp的了解扩展到极端耐寒物种提供了机会，并在甲虫Cucujus clavipe的情况下，比较来自北极阿拉斯加和印第安纳州的种群。阿拉斯加葫芦幼虫的冬季平均过冷点为-42oC，部分个体过冷至-57oC，印第安纳州幼虫的过冷点为-24oC。到目前为止，阿拉斯加葫芦的AFP活性是有史以来最高的，尽管AFP与其他甲虫的AFP相似。了解葫芦巴蛋白的结构/功能关系，以及提高其活性的增强剂(其他蛋白质和多元醇)，是首要关注的问题。除了AFP，还有一系列其他越冬适应(极度脱水到正常体水的1/3，多摩尔甘油浓度，滞育)导致阿拉斯加葫芦过冷点较低。正在对这些进行研究，并使用微阵列技术和其他手段来监测分子标记，首席调查人员将试图了解这些适应的整合。其他研究还涉及其他三种阿拉斯加甲虫AFP的结构/功能关系，以及昆虫类型AFP的结构/功能关系。其中两种甲虫是耐寒的，这是第一次对耐寒昆虫的AFP进行研究。首席调查员将继续筛选更多物种是否存在AFP(以确定未来研究的有趣候选物种)，并监测选定物种的微生境温度和越冬死亡率。这项研究的更广泛的影响涉及到它对1)生物教育和2)AFP在科学和技术中的应用的影响。博士后、博士和本科生以及高中教师将致力于这个项目。首席调查人员认为，综合培训(现代分子技术，如微阵列、田间生物学、耐寒生理学、蛋白质生物化学等)他们将获得在当今高度一体化和不断变化的研究环境中取得成功的关键准备。这将为生物学教学带来直接的好处。除了研究论文外，首席调查员还将把这些研究纳入综述文章、研讨会报告中。此外，由于它们是已知的最活跃的AFP，昆虫AFP(特别是来自阿拉斯加甲虫的AFP)具有巨大的应用研究潜力。例如，他们已经培育出了产生昆虫AFP的转基因植物，从而降低了植物的冰冻温度。使用阿拉斯加甲虫AFP进行类似的工作应该会更有成效。

This is a collaborative proposal by Principal Investigators at the University of Alaska-Fairbanks and Purdue University. Antifreeze proteins (AFPs) are found in a number of diverse organisms including fish, insects, spiders, mites, plant, and bacteria. While there is tremendous structural variation among these AFPs they all appear to lower the freezing point of water by a similar non-colligative mechanism. However, the insect AFPs are, by far, the most active, generally functioning to prevent freezing in freeze susceptible insects by inhibiting ice nucleating agents and innoculative freezing across the cuticle. However, a few AFP-producing insects are freeze tolerant and the function of AFPs in these is unknown. Two insect AFPs have been characterized. One type has been described from two species of beetles, but there is variation in sequence, etc., both within and between the species. Until recent research identified 18 species with AFPs, these proteins had not been reported in Alaskan or arctic insects. Certain of these have been chosen for further study, presenting the opportunity to extend understanding of insect AFPs to extremely cold tolerant species, and in the case of the beetle Cucujus clavipes ,to compare populations from arctic Alaska to those from Indiana. The mean winter supercooling point of Alaskan Cucujus larvae are -42oC, with some individuals supercooling to -57oC, while that of Indiana larvae is -24oC. AFP activity in Alaskan Cucujus is, by far, the highest ever described, even though the AFPs are similar to those of other beetles. Understanding the structure/function relationships of the Cucujus AFPs, and the enhancers (other proteins and polyols) which increase their activity, is of prime concern. In addition to AFPs, a suite of other overwintering adaptations (extreme dehydration to 1/3 normal body water, multimolar glycerol concentrations, diapause) contribute to the low supercooling points of Alaskan Cucujus. These are being studied, and using microarray technology and other means to monitor molecular markers, the Principal Investigators will attempt to understand the integration of these adaptations. Additional investigations involve the structure/function relationships of three other Alaskan beetle AFPs, and that of the insect type of AFP. Two of the beetle species are freeze tolerant and this is the first study of AFPs from freeze tolerant insects. The Principal Investigators will continue to screen additional species for the presence of AFPs (to identify interesting candidates for future study), and to monitor microhabitat temperatures and overwintering mortality of select species. The broader impact of this study concerns its effects on 1) biological education and 2) applications of AFPs in science and technology. Post-docs, PhD and undergraduate students, and high school teachers will work on this project. The Principal Investigators believe that the integrated training (in modern molecular techniques such as microarray, field biology, physiology of cold tolerance, protein biochemistry, etc.) at they will receive provides critical preparation for success in today's highlyintegrative and changing research environment. This will provide a direct benefit to teaching in biology. In addition to research papers, the Principal Investigators will incorporate these studies in review articles, symposium presentations. Also, as they are the most active AFPs known, insect AFPs (especially from Alaskan beetles) have tremendous potential for applied studies. For example, they have generated transgenic plants producing insect AFPs which lower the plant freezing temperature. Similar work using Alaskan beetle AFPs should be even more productive.