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

罗非鱼是一种对环境（pH值、盐度、污染）压力和变化具有多种承受能力的鱼类。该项目将比较两种罗非鱼，以破译鱼类（以及一般动物）应对环境压力和变化的生物机制。环境变化是全球关注的问题，可以在一系列时间尺度（日、年、年等）和空间尺度（地方、区域等）上发生。该项目开发并应用新的分子生物学方法来揭示耐压罗非鱼应对环境（特别是盐度）变化的机制，但在应激敏感罗非鱼中却缺乏这种机制。这一点尤其重要，因为罗非鱼是世界上增长最快的水产养殖商品，关于在发展中国家为增加粮食产量选择哪些品种的知识至关重要。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，该项目有助于评估罗非鱼在非本地生境中养殖的潜在入侵性。该项目通过培训研究生和本科生，包括在科学领域代表性不足的少数民族学生，来推进教育。它还向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换。此外，该项目将产生并提供分子生物学工具和方法，可用于许多其他生物学领域的研究人员。该项目验证了一个整体假设，即广盐罗非鱼与窄盐罗非鱼的不同之处在于盐度诱导对高渗透耐受性/耐盐性所必需的特定mrna和蛋白质的调节。研究的重点将是先前鉴定的渗透调节蛋白、肌醇磷酸合酶（MIPS）、肌醇单磷酸酶1 （IMAP1）和Na-K atp酶α 3亚基（NKA3a），以揭示这些基因的调节与耐盐性之间的因果关系。目的1是测定全盐罗非鱼（Oreochromis mossambicus）与同属窄盐罗非鱼O. niloticus中受盐度改变的mrna和蛋白质。这一目标包括比较盐度对mRNA和蛋白质丰度的影响，以及鉴定相关的单氨基酸变异（sav）和翻译后修饰。目的2是利用实验（增强子捕获）和生物信息学（基序搜索）相结合的方法揭示广盐罗非鱼的诱导顺式元件。目的3是通过亲和、功能互补和质谱方法纯化激活罗非鱼IMPA1和MIPS基因中渗透/盐度响应增强子的转录因子。目的4包括在罗非鱼细胞中进行基因组编辑，以确定MIPS和IMPA1基因以及NKA3a SAV A428？是维持细胞高渗透性所必需的。对细胞表型的影响将通过细胞形态学/增殖试验、转录组学、蛋白质组学和代谢组学进行评估。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.