Finding the Genes That Matter: Profiling Gene Expression in Strongylocentrotid Sea Urchins with Different Biogeographic and Temperature Distributions

寻找重要的基因：分析不同生物地理和温度分布的 Strongylocentrotid 海胆的基因表达

• 批准号: 0425107
• 负责人: Gretchen Hofmann
• 金额: --
• 依托单位: University of California-Santa Barbara
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-15 至 2009-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0425107&HistoricalAwards=false
• 关键词: Finding; Genes; Matter; Profiling; Gene

The goal of this project is: (1) to test the hypothesis that temperature contributes to setting species distribution patterns in marine ecosystems and (2) to employ genomic approaches and other molecular techniques to assess changes in gene expression in congeneric sea urchins that are adapted to different temperatures and have different biogeographical distributions in nature. The project will use four congeneric species of temperate sea urchin (genus Strongylocentrotus) as study organisms: S. purpuratus, S. droebachiensis, S. franciscanus, and S. pallidus. These marine invertebrates are ideal study organisms for this study because (1) the congeners are closely-related yet have different ecological temperature ranges that facilitate comparative studies, (2) it is possible to work on early life history stages as well adults with relative ease and the embryos have intraspecific differences in response to temperature, and (3) there are significant, readily-available molecular resources for the purple sea urchin, S. purpuratus, as a result of the Sea Urchin Genome Project. Gene expression profiles will be assessed in order to address how the differentially thermally adapted sea urchin species respond to ecologically relevant temperatures at the genomic level. The strategy for the genomics approach entails using DNA macroarrays to screen for differentially expressed genes that will then be analyzed using other genomics techniques -- DNA microarrays and real time PCR. The objectives of the research component are focused around the following three questions: Question 1: Are early life history stages of strongylocentrotid sea urchins differentially sensitive to temperature with respect to patterns gene expression and thermotolerance? This aspect of the project will test whether embryos of the sea urchin congener are differentially sensitive to temperature. Embryos will be raised using standard procedures and thermotolerance of different stages will be assessed in thermal trials. Different embryo stages for each species will be exposed to a range of temperatures and then sampled in order to profile temperature-dependent gene expression in the embryos. Gene expression patterns in larval stage sea urchins will be assessed using the genomics strategy where macroarrays are used to screen for gene expression patterns. Question 2: Do adults display differential responses to temperature as detected at the genomic scale? Adult specimens from each species will be acclimated to the same temperatures and molecular techniques will be used to assess differential gene expression patterns. Congeneric adults will be acclimated in aquaria at 5 and 15 C for 4 weeks. Samples (dissected tube feet) will be analyzed for expression pattern variations in the acclimated sea urchins immediately following acclimation and in the 2 h heat challenge samples. cDNA macroarray, followed by DNA microarray experiments on targeted genes, will be used to compare the expression in the sea urchins. Question 3: How do temperature-dependent patterns of gene expression vary on a latitudinal scale in two Strongylocentrotus congeners with distinctly different distributions in nature? The hypothesis that differential gene expression will be tested, i.e. variable transcript abundance and species, correlates with the different distributions of the sea urchin congeners. Specimens will be collected at different points in the biogeographic range and the transcript profile, as measured using DNA microarrays, will be assessed and correlated to thermal range of the species. Broader Impacts & Education: The ultimate contribution of this study is to bring techniques of functional genomics to bear on questions in marine systems. The results will provide insight into how marine organisms function in the marine environment, and how they respond to variation in their physical environment. Regarding education and training, in addition to the standard training of undergraduates and graduate students in biological research, the PI will collaborate with Oceans Alive!, an on-going educational outreach program at UCSB's Marine Science Institute and the Research Experience and Education Facility (REEF). REEF is a learning lab committed to formal and informal educational activities for K-12 students and the public. The PI.s lab will work with the REEF manager and the MSI educational director to develop interpretive displays of local intertidal organisms and educational and training materials for REEF docents.

该项目的目标是：（1）检验温度有助于确定海洋生态系统中物种分布模式的假设，（2）采用基因组方法和其他分子技术评估适应不同温度并在自然界中具有不同地理分布的同类海胆的基因表达变化。该项目将使用四种同属的温带海胆（球海胆属）作为研究生物：S。purpuratus，S. droebachiensis，S. franciscanus和S.苍白球这些海洋无脊椎动物是这项研究的理想研究生物，因为（1）同源物是密切相关的，但有不同的生态温度范围，有利于比较研究，（2）可以相对容易地研究早期生活史阶段以及成年人，胚胎对温度的反应具有种内差异，（3）有显着的，紫海胆S.这是海胆基因组计划的成果。 将评估基因表达谱，以解决差异热适应海胆物种如何在基因组水平上对生态相关温度作出反应。基因组学方法的策略需要使用DNA微阵列来筛选差异表达的基因，然后使用其他基因组学技术-- DNA微阵列和真实的时间PCR--来分析这些基因。研究部分的目标集中在以下三个问题：问题1：是否早期的生活史阶段的圆海胆差异敏感的温度模式，基因表达和耐热性？该项目的这一方面将测试海胆同源物的胚胎是否对温度有不同的敏感性。胚胎将使用标准程序培养，并在热试验中评估不同阶段的耐热性。每个物种的不同胚胎阶段将暴露于一系列温度下，然后取样，以分析胚胎中温度依赖性基因的表达。在幼虫阶段的海胆基因表达模式将使用基因组学策略，其中宏阵列用于筛选基因表达模式进行评估。 问题2：成年人在基因组尺度上对温度的反应是否存在差异？ 每个物种的成年标本将适应相同的温度，并将使用分子技术评估差异基因表达模式。同类成虫将在5和15 ℃的水族箱中驯化4周。 将分析样品（解剖的管脚）在驯化后立即驯化的海胆和2 h热挑战样品中的表达模式变化。 cDNA宏阵列，随后对靶基因进行DNA微阵列实验，将用于比较海胆中的表达。 第三题：在自然界分布明显不同的两种球海胆同源物中，基因表达的温度依赖模式在纬度尺度上是如何变化的？将测试差异基因表达的假设，即可变的转录本丰度和物种，与海胆同源物的不同分布相关。 将在微电泳范围内的不同点收集标本，并评估使用DNA微阵列测量的转录谱，并将其与物种的热范围相关联。 更广泛的影响教育：这项研究的最终贡献是将功能基因组学技术应用于海洋系统的问题。 研究结果将有助于深入了解海洋生物如何在海洋环境中发挥作用，以及它们如何对物理环境的变化作出反应。 在教育和培训方面，除了对本科生和研究生进行生物研究方面的标准培训外，PI还将与Oceans Alive！UCSB海洋科学研究所和研究经验和教育设施（REEF）正在进行的教育推广计划。 REEF是一个学习实验室，致力于为K-12学生和公众提供正式和非正式的教育活动。PI的实验室将与REEF经理和MSI教育总监合作，为REEF讲解员开发当地潮间生物的解释性展示以及教育和培训材料。