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

罗非鱼是一组对环境(pH、盐度、污染)压力和变化具有非常不同能力的鱼类。该项目将对两种罗非鱼进行比较，以破译鱼类(和一般动物)应对环境压力和变化的生物机制。环境变化是全球关注的问题，可以在一系列时间尺度(每日、年度、十年等)上发生。和空间尺度(地方、区域等)。该项目开发并应用了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)变化所使用的机制，但缺乏对应激敏感的罗非鱼。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，了解在发展中国家选择哪些物种以增加粮食产量至关重要。这些研究还告诉我们，这些鱼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侵袭性。该项目通过培训研究生和本科生，包括在科学界代表性不足的少数族裔学生，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，该项目将产生和提供可供研究人员在许多其他生物学领域使用的分子生物学工具和方法。该项目测试了这样的总体假设，即真盐罗非鱼通过盐度诱导对高渗透/耐盐性所必需的特定mRNA和蛋白质的调节而不同于它们的狭盐同系物。重点将集中在先前发现的渗透调节蛋白，肌醇磷酸合成酶(MIPS)，肌醇单磷酸酶1(IMAP1)和Na-K-ATPaseα3亚单位(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是比较同属狭盐罗非鱼(O.niloticus)，确定受盐度影响的罗非鱼(Oreochromis Mossambius)的mRNAs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，以及识别相关的单一氨基酸变体(SAV)和翻译后修饰。目标2是利用实验(增强子捕获)和生物信息学(基序搜索)相结合的方法揭示真盐基罗非鱼中可诱导的顺式元件。目的3通过亲和力、功能互补和质谱学等方法纯化罗非鱼IMPA1和MIPS基因中能激活渗透压/盐度反应增强子的转录因子。目标4包括罗非鱼细胞的基因组编辑，以确定MIPS和IMPA1基因以及NKA3a SAVA428？S是否是维持细胞高渗透压耐受所必需的。对细胞表型的影响将通过细胞形态/增殖分析、转录组学、蛋白质组学和代谢组学来评估。目标5是在整个罗非鱼中执行由目标4通知的目标的基因组编辑。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.