中华鳖血液细胞的分层鉴定与转录组比较分析

Stratified Identification and Bulk Transcriptome Comparison of Blood Cells of Pelodiscus sinensis

本研究以中华鳖(Pelodiscus sinensis)为研究对象,利用Percoll分离法,成功从Percoll分离液不同密度层(浓度大于60%、20%~40%和50%~60%)中分别获得血液红细胞、淋巴细胞和血栓细胞。本研究对中华鳖血液细胞类型进行了更精确的鉴别和描述,并展示这3种类型细胞的转录组中重要标记基因的表达。转录组结果显示,淋巴细胞与红细胞间、血栓细胞与红细胞间以及血栓细胞与淋巴细胞间,分别筛选出1701、1570和1280个差异表达基因。GO富集分析显示,红细胞上调基因具有溶菌酶活性和转移酶活性,参与活性氧代谢过程的调控;淋巴细胞上调差异基因与细胞因子活性有关,参与免疫反应调节细胞表面受体信号过程和对病毒的响应;血栓细胞差异基因主要参与髓样白细胞活化、粒细胞激活和防御反应。KEGG富集分析显示,淋巴细胞与红细胞的差异基因富集到NF-κB信号、EB病毒感染、T细胞受体信号等通路;血栓细胞与红细胞的差异基因富集到血小板激活和造血细胞谱系等;淋巴细胞和血栓细胞的差异基因富集于IL-17信号通路、病毒蛋白与细胞因子及细胞因子受体的相互作用等过程。同时,血液分层转录组的聚类分析结果为中华鳖血液红细胞、淋巴细胞和血栓细胞提供了潜在标记基因。

[Objectives]Transcriptome sequencing was performed on the samples collected after blood discontinuous density gradient centrifugation to explore the key functions of the blood cells of Chinese Soft-shelled Turtle Pelodiscus sinensis and to find out the marker genes related to immune function.[Methods]Blood samples of P.sinensis were stained with DQ to further characterize and describe the cell types.We used Percoll separation method to isolate erythrocytes,lymphocytes and thrombocytes of P.sinensis from whole blood with densities greater than 60%,20%﹣40%and 50﹣60%for bulk transcriptome sequencing,with 3 biological replicates per sample.[Results]DQ staining showed the morphological characteristics of 7 blood cells of P.sinensis(Fig.1).Venn diagram analysis results showed that coexpressed and specifically expressed genes were screened based on expression levels between the two groups,among which 274,628 and 295 specific genes were expressed in erythrocytes,lymphocytes and thrombocytes,respectively(Fig.2a).1701,1570 and 1280 differentially expressed genes(DEGs)were screened between lymphocyte and erythrocyte groups,between thrombocyte and erythrocyte groups,and between thrombocyte and lymphocyte groups,respectively(P–adjust<0.001)(Fig.2b).GO enrichment showed that the up-regulated gene of erythrocytes had lysozyme activity and transferase activity,and was involved in the regulation of active oxygen metabolism(Fig.3a).The up-regulated differential genes of lymphocytes were related to cytokine activity,and participated in the immune response to regulate the signaling process of cell surface receptors and the response to viruses(Fig.3b).The up-regulated differential genes of thrombocytes were mainly involved in myeloid leukocyte activation,granulocyte activation and defense response(Fig.3c).KEGG enrichment showed that the differential genes of lymphocytes and erythrocytes were enriched to NF-κB signaling,EB virus infection,T cell receptor signaling pathway,etc(Fig.4a).The differential genes of thrombocytes and erythrocytes were enriched to platelet activation and hematopoietic cell lineage(Fig.4b).Differential genes of thrombocytes and lymphocytes were enriched to the IL-17 signaling pathway,the interaction of viral protein with cytokines and cytokine receptors in lymphocytes and thrombocytes cells(Fig.4c).The cluster analysis results of stratified blood bulk transcriptome revealed important potential marker genes of three types of cells screened from single-cell transcriptome(Fig.5).[Conclusion]We further explored the function of blood cells of P.sinensis turtle by using discontinuous density gradient centrifugation combined with bulk transcriptome sequencing.The high expression of specific genes in the three cell types provided a basis for cell marker gene screening and antibody development of P.sinensis.