基于单细胞转录组测序技术筛选蟾蜍耳后腺免疫修复基因

Screening of Retroauricular Gland Immune Repair Genes by scRNA-seq in Bufo bufo gargarizans Cantor

中华大蟾蜍是我国传统药源动物,是名贵中药材蟾酥的基原物种之一。为筛选中华大蟾蜍耳后腺免疫修复基因,本试验采用Illumina测序平台对成年中华大蟾蜍耳后腺组织样本进行单细胞转录组测序(scRNA-seq),完成scRNA-seq文库构建后进行细胞亚群分类,对细胞亚群中上调表达基因和免疫修复相关基因进行分析,并通过实时荧光定量PCR(qRT-PCR)对候选基因进行初步筛选与验证。结果表明,蟾蜍耳后腺组织中用于文库构建和双端测序的细胞数量为8414个,每个细胞的平均读数和中位基因数为46326和734,可用于进一步分析的高质量细胞数为7687个。聚类分析得到12个细胞亚群,并推测Cluster 10是来源于结缔组织的浆细胞。通过GO和KEGG分析对Cluster 10中的217个上调表达基因进行分析,鉴定出多个参与机体免疫的生物过程和通路,包括淋巴细胞活化、淋巴细胞分化及白细胞活化等生物过程和细胞受体信号通路、Fc epsilon RI信号通路及趋化因子信号通路等免疫相关通路。从Cluster 10的差异基因中筛选出MYO1F基因和CYBB基因为中华大蟾蜍耳后腺免疫修复基因,并通过qRT-PCR对两基因进行验证,推测对免疫修复具有正向调节作用。本研究结果可为后续挖掘蟾蜍耳后腺免疫修复基因、研究蟾蜍耳后腺采酥后的修复机制提供理论依据,为蟾蜍耳后腺单细胞组学研究提供参考信息。

Bufo bufo gargarizans Cantor is a kind of traditional medicinal animal and one of the original species of BUFONIS VENENUM.In order to explore the genes related to immune repair of its retroauricualr gland,single-cell RNA-sequencing(scRNA-seq)was carried out with adult B.bufo gargarizan retroauricular gland tissue samples using the Illumina sequencing technology.Cell subgroups were classified after construction of the scRNA-seq library,the up-regulated genes and those related to immune repair in the cell subgroups were analyzed,and real-time fluorescence quantitative PCR(qRT-PCR)was used to screen the candidate genes initially.The results showed that the retroauricular gland of a toad had 8414 cells used for library con-struction and paired-end sequencing.The average reads and median gene number per cell were 46326 and 734,respectively,and the number of high-quality cell available for additional analysis was 7687.Twelve cell subgroup were obtained through cluster analysis,and it was hypothesized that Cluster 10 was plasma cell origi-nated from connective tissue.The 217 up-regulated genes in Cluster 10 were analyzed by GO and KEGG analy-ses,and a number of biological processes and pathway related to body immunity were identified,including bi-ological processes such as lymphocyte activation,lymphocyte differentiation and leukocyte activation,and im-mune related pathways such as cell receptor signaling pathway,Fc epsilon RI signaling pathway and chemo-kine signaling pathway.The MYO1F and CYBB genes were select from the differential genes of Cluster 10 as the immune repair genes of the retroauricular gland in B.bufo gargarizans,which were verified by qRT-PCR and speculated to have positive regulatory relationships with immune repair.The findings of this study could provide theoretical references for the subsequent excavation of immune repair genes and the repair mechanism research,and provide reference information for single cell genomics study in toad retroauricular gland.