杨平, 刘洁, 陈再忠, 陆颖. 2024: 天子蓝盘丽鱼皮肤组织的单细胞转录组测序. 南方农业学报, 55(3): 601-610. DOI: 10.3969/j.issn.2095-1191.2024.03.002
引用本文: 杨平, 刘洁, 陈再忠, 陆颖. 2024: 天子蓝盘丽鱼皮肤组织的单细胞转录组测序. 南方农业学报, 55(3): 601-610. DOI: 10.3969/j.issn.2095-1191.2024.03.002
YANG Ping, LIU Jie, CHEN Zai-zhong, LU Ying. 2024: Single-cell transcriptome sequencing of skin tissue in blue discus fish (Symphysodon spp.). Journal of Southern Agriculture, 55(3): 601-610. DOI: 10.3969/j.issn.2095-1191.2024.03.002
Citation: YANG Ping, LIU Jie, CHEN Zai-zhong, LU Ying. 2024: Single-cell transcriptome sequencing of skin tissue in blue discus fish (Symphysodon spp.). Journal of Southern Agriculture, 55(3): 601-610. DOI: 10.3969/j.issn.2095-1191.2024.03.002

天子蓝盘丽鱼皮肤组织的单细胞转录组测序

Single-cell transcriptome sequencing of skin tissue in blue discus fish (Symphysodon spp.)

  • 摘要: 【目的】通过单细胞转录组测序比较天子蓝盘丽鱼和野生阿莲卡盘丽鱼皮肤组织中与体色形成相关的基因表达模式,为深入研究鱼类体色及良种选育提供数据基础,也为盘丽鱼或其他水产鱼类进行单细胞转录组研究提供技术借鉴。【方法】利用单细胞转录组测序技术对天子蓝(人工选育)和阿莲卡(野生型)2种盘丽鱼的皮肤组织进行转录组测序,经fastp质控过滤后,通过Cell Ranger比对参考基因组生成单细胞表达矩阵文件,使用Seurat进行降维聚类后构建转录组图谱,筛选出细胞类型识别与鉴定的标记基因;同时以阿莲卡盘丽鱼为对照,通过DEGseq筛选出2个样本间的差异表达基因(DEGs),并进行GO功能注释分析和KEGG信号通路富集分析。【结果】经单细胞转录组测序,从天子蓝、阿莲卡盘丽鱼样本中获得的原始序列(Raw reads)分别为815237286和728886552条;将2个样本整合后共捕获17035个细胞,经过滤及降维聚类后得到18个细胞簇(C0~C17),参考已知和潜在的标记基因,被注释为13种细胞类型及其亚型,基本涵盖了鱼类皮肤组织中的大部分细胞类型。基于伪批量(Pseudo-bulk)处理,从2个样本间筛选出20个DEGs(8个在天子蓝盘丽鱼皮肤组织中高表达,12个在阿莲卡盘丽鱼皮肤组织中高表达),主要注释到与气体传递、氧化代谢相关的GO功能条目。在单细胞维度,选择黑色素细胞和虹彩细胞2个细胞集群进行样本间差异分析,结果显示:在黑色素细胞中存在8个DEGs,主要富集于热应答、未折叠蛋白结合、生长因子活性、金属离子结合等信号通路。在虹彩细胞中存在25个DEGs,KEGG信号通路富集分析发现主要富集于凋亡、沙门氏菌感染和MAPK等信号通路,涉及细胞生存、应激响应和信号传导等功能。【结论】TSPAN3A、PTGES、ATF3、JUNK、GADD45GA、HSP70L、FOSAB等基因在调节天子蓝盘丽鱼皮肤体色、色素合成与沉着等方面发挥重要作用,而黑色素合成不活跃可能是导致天子蓝盘丽鱼体色较阿莲卡盘丽色更加鲜艳和绚丽的主要原因之一。

     

    Abstract: 【Objective】To profile and compare the expression patterns of the genes involved in formation of body color between the skin tissues of blue discus fish and wild Alenquer discus fish through a single-cell transcriptome sequencing, which provided the data basis for further study of body color and breeding, as well as the technical reference for the single-cell transcriptome studies in discus fish and other fishes. 【Method】Using the single-cell transcriptome sequencing technology, transcriptome sequencing was performed on the skin tissues of blue discus fish(bred strain) and Alenquer discus fish (wild type). The expression matrix of single-cell data was generated by Cell Ranger alignment against reference genomes after fastp quality control filtration, which was used for dimensionality reduction clustering to construct transcriptome atlas by Seurat and indentification of cell types referencing the marker genes. The differentially expressed genes (DEGs) between the two samples were identified by DEGseq, which was annotated through the GO functional analysis and KEGG pathway enrichment. 【Result】Through single-cell transcriptome sequencing, up to 815237286 and 728886552 raw reads were generated in blue discus and Alenquer discus, respectively. A total of 17035 cells were captured after the integration of the two samples’ data, which built 18 clusters(C0-C17) after filtering and dimensionality reduction. Referred to known and potential marker genes, these clusters were divided into 13 cell types and their subtypes, which encompassed most of the cell types in fish skin. Based on pseudo-bulk treatment, 20 DEGs were detected from the 2 samples (8 were highly expressed in blue discus and 12 were highly expressed in Alenquer discus), mainly annotated to GO functional terms of gas transport and oxidative metabolism. In the single-cell dimension, two cell clusters of melanocytes and iridophore were selected for inter-sample difference analysis, which detected eight DEGs in melanocytes, involving in response to heat, cytoplasm, unfolded protein binding, growth factor activity and metal ion binding. Twentyfive DEGs were found in iridophore, which were mainly employed in the signaling pathwasy,such as apoptosis, Salmonella infection and MAPK, cell survival, stress response and signaling. 【Conclusion】TSPAN3A, PTGES, ATF3, JUNK, GADD45GA, HSP70L and FOSAB genes play an important role in regulation of pigment biosynthesis and pigmentation of blue discus fish skin. The inactive synthesis of melanin probably results in more vivid and gorgeous body color in blue discus than Alenquer discus.

     

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