盐度驯化影响虹鳟鳞组织基因表达的转录组分析

Analysis of the transcriptomic response to salinity acclimation in the scale of rainbow trout (Oncorhynchus mykiss)

中文关键词: 虹鳟  鳞  盐度驯化  转录组测序  实时荧光定量PCR

英文关键词: Oncorhynchus mykiss  Scales  Salinity acclimation  RNA sequencing  Quantitative real-time PCR

中文摘要:

      合理选择盐度驯化方式是目前虹鳟(Oncorhynchus mykiss)养殖生产所要解决的重要问题之一。本研究通过分析盐度驯化对虹鳟幼鱼外骨骼(鳞组织)中基因表达水平的影响，重点探讨了盐度对鱼类骨代谢的影响机制。首先，分别采集海水(盐度28)驯化7 d和常规淡水养殖(对照)条件下的虹鳟鳞组织，采用Illumina HiSeq 4000测序平台进行转录组测序(RNA-Seq)。以log2|fold change|≥1且P<0.05作为显著差异表达基因(DEGs)筛选条件，共筛选出1714个DEGs，其中，484个基因显著上调，1230个基因显著下调。GO功能注释分析结果显示，上述DEGs主要被注释在细胞膜、细胞质、细胞核、运输、信号转导、金属离子结合和ATP结合等功能中。KEGG通路富集分析结果显示，DEGs在氧化磷酸化、药物代谢–细胞色素P450、蛋白酶体、p53信号通路和心肌收缩等通路中显著富集。利用实时荧光定量PCR (RT-qPCR)对随机选取的8个DEGs的表达量进行验证，结果显示，RT-qPCR与RNA-Seq结果一致，表明RNA-Seq数据可靠。结果表明，以4/d的盐度提升速率对虹鳟进行盐度驯化，对其Mmp-2、Mmp-9、Acp5b、Alpl、Osteocalcin、OPG和Col12a1等骨代谢相关基因及NF-kB、MAPK-(JNK、p38、ERK1/2和STAT3)、Wnt/β-catenin、BMP/Smads和OPG-RANK-RANKL等骨代谢相关信号通路影响不显著，说明本研究所采用的驯化模式较为合理。本研究结果可为虹鳟的养殖生产实践提供参考，所获得的基因信息、功能注释和通路富集信息可为探究硬骨鱼类骨代谢的调控机理及其应对环境变化的演化规律提供新视角。

英文摘要:

      The reasonable selection of salinity acclimation is one of the most important problems in Oncorhynchus mykiss culture. Calcium metabolism in fish varies with changing environmental salinity, and is a crucial component of salt metabolism. The scale compartment constitutes a significant, readily accessible calcium source in fish, as it can contain up to 20% of the body´s total calcium. In terms of complexity, scales resemble bone better than cultured osteoblast or osteoclast cell lines. Therefore, scales are ideal models for calcium metabolism and bone research. To provide more reliable data to decipher the bone metabolism at the molecular level, an analysis of the transcriptomic response to salinity acclimation was performed on rainbow trout scales. Fish were subjected to seawater (salinity 28) acclimation or freshwater maintenance for seven days. RNA sequencing (RNA-Seq) was performed using the Illumina HiSeq 4000 sequencing platform. By setting the screening conditions for the significant differentially expressed genes (DEGs) as log2|fold change|≥1 and P<0.05, 1714 DEGs were identified, whereof 484 and 1230 were significantly upregulated and downregulated, respectively. Gene Ontology function annotation analysis showed that the DEGs were primarily annotated in biological functions such as cell membrane, cytoplasm, nucleus, transportation, signal transduction, metal ion binding, and ATP binding. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the DEGs were significantly enriched in pathways such as oxidative phosphorylation, drug metabolism-cytochrome P450, proteasome, p53 signaling pathway, and myocardial contraction. Furthermore, quantitative real-time PCR (RT-qPCR) was used to examine the expression of eight randomly-selected DEGs, and the results of the RT-qPCR and RNA-Seq were consistent, indicating the reliability of the RNA-Seq data. Unigene in rainbow trout scales are predominantly enriched in bone metabolism-related pathways, such as the MAPK, Wnt, and calcium signaling pathways, indicating that scales can be used as a model for bone metabolism research. The results of this study showed that the salinity acclimation of O. mykiss was carried out at a rate of salinity 4 per day; bone metabolism-related genes, such as Mmp-2, Mmp-9, Acp5b, Alpl, Osteocalcin, OPG and Col12a1; and bone metabolism-related signaling pathways, such as NF-kB, MAPK-(JNK, p38, ERK1/2, STAT3), Wnt/β-catenin, BMP/Smads, and OPG-RANK-RANKL, did not have a significant influence, indicating that the acclimation model adopted in this study was reasonable. The results could help clarify the regulatory mechanism of O. mykiss bone metabolism in response to altered salinity, and lay a theoretical foundation for aquaculture industry development.