NSF-IOS-BSF: Biochemical and genetic basis of salinity tolerance in tilapia

罗非鱼是一组具有非常不同的能力来承受环境（pH值，盐度，污染）压力和变化的鱼类。该项目将比较两种罗非鱼，以破译鱼类（和一般动物）应对环境压力和变化的生物机制。环境变化是一个全球性的问题，可以发生在一系列的时间尺度（每日，每年，十年等）。和空间尺度（地方、区域等）。该项目开发和应用新的分子生物学方法，以揭示耐胁迫罗非鱼（但缺乏对胁迫敏感的罗非鱼）应对环境（特别是盐度）变化的机制。这一点尤其重要，因为罗非鱼是世界范围内增长最快的水产养殖商品，了解发展中国家选择哪些物种增加粮食产量至关重要。这些研究还告诉我们这些鱼类以及其他动物适应环境变化所需的进化时间尺度。此外，该项目还有助于评估在非本地栖息地养殖罗非鱼的潜在入侵性。该项目通过培训研究生和本科生，包括理科人数不足的少数民族学生，促进教育。它还为K-12学生和教育工作者，水产养殖生产者和保护组织提供外展服务。它包括在非洲和以色列的国际合作和学生交流。此外，该项目还将产生和提供可供生物学许多其他领域研究人员使用的分子生物学工具和方法，该项目将检验一个总体假设，即广盐罗非鱼与其狭盐性同类鱼的不同之处在于，盐诱导调节特定mRNA和蛋白质，而这些mRNA和蛋白质是高耐盐性/耐盐性所必需的。重点将是以前确定的盐调节蛋白，肌醇磷酸合酶（MIPS），肌醇单磷酸酶1（IMAP1），和钠钾ATP酶α 3亚基（NKA3a），揭示这些基因的调节和耐盐性之间的因果关系。目的一是确定广盐性罗非鱼（Oreochromis mossambicus）与同属的狭盐性罗非鱼（O。尼罗河。这一目标包括比较盐度对mRNA和蛋白质丰度的影响，并鉴定相关的单氨基酸变体（SAVs）和翻译后修饰。目的二是利用实验（增强子捕获）和生物信息学（模体搜索）相结合的方法揭示广盐罗非鱼中可诱导的顺式元件。目的3：利用亲和层析、功能互补和质谱分析等方法，从罗非鱼IMPA 1和MIPS基因中分离出激活罗非鱼抗盐/耐盐增强子的转录因子。目的4包括在罗非鱼细胞中进行基因组编辑，以确定MIPS和IMPA 1基因以及NKA 3a SAV A428？是维持细胞高耐药性所必需的。将通过细胞形态学/增殖试验、转录组学、蛋白质组学和代谢组学评估对细胞表型的影响。目标5是在整个罗非鱼中对目标4所告知的靶标进行基因组编辑。将分析对罗非鱼耐盐性、形态、行为、生理和分子表型（转录组、蛋白质组、代谢组）的影响。

Tilapias are a group of fish with very diverse abilities to tolerate environmental (pH, salinity, pollution) stress and change. This project will compare two tilapia species to decipher the biological mechanisms used by fish (and animals in general) for coping with environmental stress and change. Environmental change represents a global concern and can occur over a range of time scales (daily, annual, decadal etc.) and spatial scales (local, regional, etc.). This project develops and applies novel molecular biology approaches for revealing the mechanisms used by stress-tolerant tilapia, but lacking in stress-sensitive tilapia, for coping with environmental (in particular, salinity) change. This is especially important because tilapia are the most rapidly growing aquaculture commodity world-wide and knowledge about which species to select for increased food production in the developing world is critical. These studies also inform us about the evolutionary time scales needed for these fish, as well as other animals, to adapt to environmental changes. In addition, the project facilitates assessment of the potential for invasiveness if tilapia are cultured in non-native habitats. This project advances education by training of graduate and undergraduate students, including students from minorities that are underrepresented in the sciences. It also provides outreach to K-12 students and educators, aquaculture producers, and conservation organizations. It includes international collaborations and student exchange in Africa and Israel. In addition, this project will generate and make available molecular biology tools and approaches that can be used by researchers in many other areas of biology.This project tests the overall hypothesis that euryhaline tilapia differ from their stenohaline congeners by salinity-induced regulation of specific mRNAs and proteins that are necessary for high osmotolerance/ salinity tolerance. The focus will be on previously identified osmoregulatory proteins, myo-inositol phosphate synthase (MIPS), inositol monophosphatase 1 (IMAP1), and the Na-K ATPase alpha 3 subunit (NKA3a), to reveal causality between the regulation of these genes and salinity tolerance. Objective 1 is to determine mRNAs and proteins that are altered by salinity in euryhaline tilapia (Oreochromis mossambicus) compared to a congeneric stenohaline species, O. niloticus. This objective includes comparison of salinity effects on mRNA and protein abundances and identification of relevant single amino acid variants (SAVs) and post-translational modifications. Objective 2 is to reveal inducible cis-elements in euryhaline tilapia using a combination of experimental (enhancer trapping) and bioinformatics (motif searching) approaches. Objective 3 is to purify the transcription factor that activates the tilapia osmotically/salinity responsive enhancer in IMPA1 and MIPS genes by affinity-based, functional complementation, and mass spectrometry approaches. Objective 4 comprises genome editing in tilapia cells to determine whether MIPS and IMPA1 genes, and the NKA3a SAV A428?¨S are necessary for maintaining high cellular osmotolerance. Effects on cellular phenotype will be assessed by cell morphology/ proliferation assays, transcriptomics, proteomics, and metabolomics. Objective 5 is to perform genome editing of a target informed by Objective 4 in whole tilapia. Effects on tilapia salinity tolerance, morphology, behavior, physiology and molecular phenotypes (transcriptome, proteome, metabolome) will be analyzed.