Comprehensive Analyses of Antifreeze Capacities in Arctic and Northern Cods at the Protein and Encoded Levels, and the Molecular of the Cod Antifreeze Glycoprotein Gene

鳕科（Gadidae）在北极和北方冷温带海洋中约有24种鳕鱼，其中包括许多商业上最重要的鳕鱼。其中五个物种已知具有抗冻糖蛋白（AFGP），这对它们在各种冷水栖息地的生存很重要。大多数鳕鱼AFGP的研究是在1975-1985年期间进行的，并提供了关于蛋白质结构和AFGP水平的季节性变化的基本信息，在一些已知的五个AFGP轴承鳕鱼。没有其他可能遇到冷冻条件的鳕鱼已经检查了可能存在的抗冻功能，没有任何信息在分子水平上的鳕鱼AFGP功能，直到1997年，当第一个AFGP基因序列和结构从北极鳕鱼Boreogadus色达报道了高AFGP编码能力在这个最北的物种。因此，不同鳕鱼种的AFGP系统远未被彻底了解。本项目建议使用现代技术对不同gadids的AFGP系统进行全面的表征和分析。总体目标是评估各自的防冻能力在蛋白质（AFGP蛋白质的结构和生产）和分子（AFGP基因和基因家族及其转录）水平，以及如何这些涉及到他们的不同的栖息地，跨越一系列的纬度和环境的严重性所施加的差异生理需求。鳕鱼的AFGP系统将在鳕鱼物种之间进行对比，并与南极对应物南极背索线虫的组成性高容量进行对比，南极背索线虫是不相关的鲈形鱼类，其在世界上最热稳定和最冷的海洋沃茨中生存的AFGP几乎相同。抗冻剂研究领域的一个关键问题是这些新的抗冻蛋白来自哪里。研究人员先前已经确定了产生南极鱼AFGP基因的进化起源和分子机制，该基因在这些鱼类在寒冷的南极沃茨的生存和生物成功中发挥了至关重要的作用。通过分子分析，该项目的第二个主要目标是破译鳕鱼AFGP基因的起源和进化历史，这导致了两个不相关的鱼类群体中相似AFGP的显着“两极”进化趋同。十种不同的海洋gadid物种，其中一种包括两个遗传上不同的种群，将被检查。具体的研究领域包括：（1）比较分析不同纬度和生境的鳕鱼种之间和种内的血液AFGP水平、蛋白质结构和异质性，以确定环境严酷性对抗冻剂产生的影响;（2）AFGP周年生产周期的确定（AFGP蛋白水平和mRNA的转录）的三个高纬度鳕鱼和它们的关系，在特定的环境条件下，他们的栖息地的时间变化;（3）通过分析其AFGP基因序列的复杂性、结构、这些基因在基因组中的组织以及基因家族的大小来确定不同鳕鱼中AFGP生产的编码能力;（4）表征AFGP表达在不同组织和非脉管流体隔室中的分布，以确定鳕鱼中所有可能的抗冻防线;（5）通过对鳕鱼（6种分布于北方的不同种类）的血液抗冻活性和基因组中AFGP样序列的检测，确定其是否具有抗冻蛋白;（6）破解鳕鱼AFGP基因的起源和进化过程。

The twenty-four or so cod species of the Arctic and northern cool-temperate oceans belonging to the family Gadidae include many of the most commercially important cod fishes. Five of these species are known to have an antifreeze glycoprotein (AFGP) that is important for their survival in their various cold-water habitats. Most of the cod AFGP studies were conducted during 1975-1985, and provided basic information on the protein structure and seasonal changes in AFGP levels in some of the five known AFGP-bearing cods. None of the other cods that may encounter freezing conditions have been examined for possible presence of an antifreeze function, and no information at all on the cod AFGP function at the molecular level was available until 1997 when the first AFGP gene sequence and structure from the Arctic cod Boreogadus saida were reported with a high AFGP coding capacity in this most-northerly species. Thus the AFGP systems of the different cod species are far from being thoroughly understood. This project proposes to conduct comprehensive characterizations and analyses of the AFGP system of the different gadids using contemporary techniques. The overall goal is to assess their respective freeze-avoidance capacities at both the protein (AFGP protein structures and production) and molecular (AFGP genes and gene families and their transcription) levels, and how these relate to the differential physiological demands imposed by their diverse habitats which span a range of latitudes and environmental severity. The AFGP systems of the cods will be contrasted among the cod species, and with the constitutive high capacities in their south-polar counterparts, the Antarctic notothenioids, which are unrelated perciform fishes that make a near-identical AFGP to survive in the world's most thermally stable and coldest marine waters at the opposite pole. A key question in the field of antifreeze research is where these novel antifreeze proteins came from. The PIs have previously determined the evolutionary origin and molecular mechanism that produced the Antarctic notothenioid AFGP gene which played a crucial role in the survival and organismal success of these fishes in the frigid Antarctic waters. Through molecular analyses, the second major goal of the project is to decipher the origin and evolutionary history of the cod AFGP gene, that had led to the remarkable 'bipolar' evolutionary convergence of similar AFGPs in two unrelated fish groups.Ten different marine gadid species, one of which includes two genetically distinct populations, will be examined. Specific areas of studies include: (1) comparative analyses of blood AFGP levels, protein structure and heterogeneity between and within cod species from different latitudes and habitats to determine the influence of environmental severity on antifreeze production; (2) determination of the circannual AFGP production cycles (AFGP protein levels and transcription of mRNA) of three high-latitude cods and their relationship to the temporal variations in environmental conditions specific to each of their habitats; (3) determination of the encoded capacity for AFGP production in different cods by analyzing the complexity of their AFGP gene sequences, structures, the organization of these genes in the genome, and the size of the gene family; (4) characterization of the distribution of AFGP expression in different tissues and non-vascular fluid compartments to determine all possible lines of defense against freezing in the cods; (5) determination whether hitherto unexamined gadids (6 different northerly distributed species) have an antifreeze protein through detection for both blood antifreeze activity and presence of AFGP-like sequences in their genome; and (6) deciphering the origin and the evolutionary process of cod AFGP gene.