基于肠转录组学探究NaHCO3碱度胁迫下红罗非鱼应激响应与耐受机制

Transcriptomic analysis of stress response and tolerance mechanisms in the intestine of red tilapia(Oreochromis spp.)during NaHCO3 exposure

为探究红罗非鱼(Oreochromisspp.)对NaHCO3碱度胁迫的应激响应机制,本研究以初始体重为(73.43±1.62)g的红罗非鱼作为研究对象,进行40 d NaHCO3碱度胁迫,比较NaHCO3碱度胁迫组[CA,(35.51±0.17)mmol/L]和淡水对照组[Con,(1.75±0.08)mmol/L]红罗非鱼血清生理参数、肠组织学和肠转录组差异。结果显示,红罗非鱼血清过氧化氢酶(CAT)活性与总抗氧化能力(TAOC)、丙二醛(MDA)、血氨和尿素氮(BUN)水平在碱度胁迫下显著增加(P<0.05);碱度胁迫导致红罗非鱼肠绒毛缩短和肠上皮损伤等现象的发生;肠转录组学分析共获得2853个差异表达基因,其中上调基因1674个,下调基因1179个。富集分析揭示了红罗非鱼肠组织响应碱胁迫的关键通路(内吞作用、氨基酸生物合成和IgA生成相关肠道免疫网络等),这些途径主要涉及物质跨膜运输、能量代谢和免疫响应等生物学过程。qRT-PCR检测验证12个碱应激响应关键基因转录水平的表达。结果表明,碱度胁迫会损伤红罗非鱼肠道组织结构,并造成其氧化应激;肠道运输、代谢和免疫响应关键基因表达的变化是红罗非鱼耐受高碱度环境的重要策略,本研究结果能够为阐释红罗非鱼对环境碱度的适应性调节机制提供理论参考。

The diminishing availability of freshwater resources in recent years has led to a decrease in suitable areas for freshwater aquaculture,which in turn has prompted the use of saline-alkaline water to meet the growing demands.However,the limited application of saline-alkaline water,threatened by the presence of a single species in saline-alkaline aquaculture,notably impedes the development of saline-alkaline aquaculture.A comprehensive understanding of their physiological and molecular mechanisms of salt-alkaline tolerance is essential to cultivate species suitable for saline-alkaline aquaculture.Red tilapia(Oreochromis spp.)has good salinity tolerance;however,the metabolic response of red tilapia under an alkaline environment remains largely unclear.In this study,we compared serum physiological parameters,intestinal histology,and transcriptome in red tilapia between an alkalinity stress group[CA,(35.51±0.17)mmol/L]and a freshwater control group[Con,(1.75±0.08)mmol/L].Exposure to the alkalinity condition for 40 d resulted in increased serum catalase(CAT)activity,total antioxidant capacity(TAOC),malondialdehyde(MDA),ammonia,and urea nitrogen(BUN)levels(P<0.05),indicating an imbalance in ammonia excretion and antioxidant defense occurred in red tilapia under alkalinity stress;notable damage to intestinal fluff,thinning of the intestinal muscle layer,and damage to intestinal epithelial cells were also observed in the CA group,suggesting that alkalinity stress may disrupt normal gut physiological function.To investigate potential regulatory mechanisms associated with the observed biochemical and morphological alterations,we conducted a transcriptome analysis.Principal component analysis(PCA)revealed a clear separation of the samples from each group,suggesting high-quality data.Based on a log2(fold change)of≥1 or≤−1 and P<0.05,we identified a total of 2853 differentially expressed genes(CA vs.Con),including 1674 upregulated and 1179 downregulated genes.A total of 234 Gene Ontology(GO)items were found to be significantly enriched,such as signal transduction,transmembrane transport,and membrane.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis revealed 112 key pathways in the intestine of red tilapia in response to alkaline stress,including endocytosis,biosynthesis of amino acids,intestinal immune network for IgA production,and NOD-like receptor signaling pathway.Gene Set Enrichment Analysis(GSEA)confirmed the activation of these four pathways under alkalinity stress.To verify the accuracy and reliability of the RNA-Seq data,a subset of 12 differentially expressed genes was chosen for qRT-PCR analysis.Correlation analysis revealed a strong linear relationship(R²=0.880)between the gene transcript level data obtained using the two methodologies,thereby validating the reliability of the transcriptome sequencing data.Overall,our results suggest that alkalinity stress may damage the intestinal structure of red tilapia and induce oxidative stress.The changes in the expression of key genes involved in intestinal transport,metabolism,and immune response are crucial strategies for red tilapia to tolerate high alkaline conditions.Our study provides essential insights into the effects of alkaline water on the health and adaptive functions of red tilapia.Furthermore,it sets a crucial basis for future research on the molecular mechanisms that govern stress responses and tolerance to saline-alkaline exposure in fish.