转录组测序解析刺参波里氏囊腔与体腔中体腔细胞对吐脏胁迫的响应差异

史伟卜; 王轶南; 王浩文; 任媛; 王庆奎; 李强

doi:10.12284/hyxb2021016

转录组测序解析刺参波里氏囊腔与体腔中体腔细胞对吐脏胁迫的响应差异

doi: 10.12284/hyxb2021016

史伟卜^{1, 2,},
王轶南¹,
王浩文¹,
任媛³,
王庆奎²,
李强^1, ,

1.
盐城工学院海洋与生物工程学院，江苏盐城 224051
2.
天津农学院水产学院，天津 300384
3.
大连海洋大学水产与生命学院，辽宁大连 116023

基金项目: 国家自然科学基金（31872544）；盐城工学院人才引进项目（XJ201725，XJ201726）。

详细信息

作者简介:
史伟卜（1995—），男，河北省邢台市人，主要从事海洋动物生理与免疫学研究。E-mail：857299973@qq.com

通讯作者:
李强，教授，主要从事海洋动物生理与免疫学研究。E-mail：fish790608@163.com

中图分类号: S917.4
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出版历程
- 收稿日期: 2020-05-23
- 修回日期: 2020-08-30
- 网络出版日期: 2021-01-21
- 刊出日期: 2021-03-02

Transcriptome analysis provides insights into the response of coelomocytes in polian vesicle and coelomic cavity of sea cucumber Apostichopus japonicus to evisceration

1.
College of Marine and Biology Engineering, Yancheng Institute of Technology, Yancheng 224051, China
2.
College of Fisheries, Tianjin Agricultural University, Tianjin 300384, China
3.
College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China

摘要

摘要: 刺参的体腔细胞大量存在于体腔液与水管系统内，广泛参与机体的营养输送、代谢以及免疫等多种功能。刺参吐脏时，体腔中体腔细胞近乎排尽，而后迅速恢复。波里氏囊是刺参吐脏后仅存的内脏器官，囊腔内体腔细胞在吐脏后也快速增加，表现出积极的响应。为研究探讨刺参吐脏后再生早期体腔细胞快速增加的作用与意义，本文分别对刺参吐脏后6 h与吐脏前波里氏囊腔和体腔中体腔细胞进行转录组测序，比较了波里氏囊腔与体腔中体腔细胞在吐脏胁迫下的基因表达变化。结果显示，波里氏囊腔内体腔细胞在吐脏前后有267个基因显著差异表达，差异基因大量富集至GO功能注释的酶催化活性亚类以及甘氨酸、丝氨酸与苏氨酸代谢等KEGG通路。体腔中体腔细胞在吐脏前后有922个基因显著差异表达，差异基因显著富集到GO功能注释的细胞黏附、生物黏附等亚类以及细胞外基质受体互作、转化生长因子-β与FoxO等KEGG通路。研究结果对于进一步研究刺参体腔细胞的功能及揭示刺参吐脏后的再生机制提供了重要基础。
- 刺参 /
- 体腔细胞 /
- 波里氏囊 /
- 吐脏 /
- 转录组
Abstract: Coelomocytes in Apostichopus japonicus, present in coelomic fluid and water-vascular system, are considered to participate in a variety of biological functions including nutrition transport, metabolism and immunity. In the process of evisceration, coelomocytes in coelom are nearly exhausted and then recovered in a short period. The polian vesicle, as the only remained internal organ after evisceration, shows positive response that coelomocytes within it increased rapidly. Coelomocytes in coelom are nearly exhausted after evisceration, and then recovered quickly. To further investigate the function and significance of the rapid increase of coelomocytes in the early stage of regeneration after evisceration, transcriptome sequencing was performed for coelomocytes in polian vesicle and coelom of A. japonicus at 6 h after evisceration and pre-evisceration, respectively. The gene expression differences of coelomocytes in polian vesicle and coelom after evisceration were analyzed compared to those of healthy A. japonicus. These results showed that 267 genes were differentially expressed in coelomocytes of polian vesicle at 6 h after evisceration, and most of these genes were enriched into the enzyme catalytic activity subclasses according to GO functional annotation and glycine, serine and threonine metabolism pathway according to KEGG pathways annotation, respectively. Additionally, 922 differential genes were significantly expressed in coelomocytes of coelom at 6 h after evisceration, and these genes were enriched into cell adhesion and biological adhesion subclasses according to GO functional annotation and ECM-receptor interaction, TGF-β signaling pathway, FoxO signaling pathway according to KEGG pathways annotation, respectively. The results provide an important basis for further functional research and the regeneration mechanism of coelomocytes after evisceration in A. japonicus.
- Apostichopus japonicus /
- coelomocytes /
- polian vesicle /
- evisceration /
- transcriptome

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图 1 PC6 h组与PC0 h组（a）及CC6 h组与CC0 h组（b）的显著差异基因分布火山图

红点代表log₂ (Fold change)>1和padj<0.05的上调基因；绿点表示log₂ (Fold change)<−1和padj<0.05的下调基因；蓝点代表没有显著差异的基因

Fig. 1 Volcano plot significantly differential expression genes distribution between PC6 h and PC0 h (a) and between CC6 h and CC0 h (b)

Red points represent up-regulated genes with log₂(Fold change)>1 and padj<0.05; green points represent down-regulated genes with log₂(Fold change)<−1 and padj<0.05; blue points represent genes with no significant difference

下载: 全尺寸图片幻灯片

图 2 GO富集分析中显著差异基因的功能分类

PC6 h和PC0 h间显著差异基因的GO分析 (a)；CC6 h与CC0 h间显著差异基因的GO分析 (b)。x轴表示GO分析中的亚类；y轴表示差异基因的数量；蓝色和绿色分别代表上调基因和下调基因

Fig. 2 Functional categorization of significantly differential expression genes in Gene Ontology

GO analysis between PC6 h and PC0 h (a); GO analysis between between CC6 h and CC0 h (b). The x-axis shows the 2nd level term of Gene Ontology; the y-axis shows the number of DEGs; the blue and brown represent up- and down-regulated genes, respectively

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图 3 显著差异基因的KEGG通路富集分析

PC6 h与PC0 h间显著差异基因的KEGG通路富集 (a)；CC6 h和CC0 h间显著差异基因的KEGG通路富集 (b)。圆点的颜色表示padj值，圆点的大小表示注释到KEGG通路上的基因数

Fig. 3 KEGG pathway enrichment of significantly differential expression genes

KEGG pathway enrichment between PC6 h and PC0 h (a); KEGG pathway enrichment between CC6 h and CC0 h (b). The color of the dot represents padj and size of the dot represents the number of DEGs mapped to the reference pathways

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图 4 qRT-PCR验证RNA-Seq结果

PC6 h和PC0 h间的RNA-Seq验证结果（a）；CC6 h和CC0 h间的RNA-Seq验证结果（b）

Fig. 4 Verification of the RNA-Seq results using the qRT-PCR method

Verification of the RNA-Seq results between PC6 h and PC0 h (a); verification of the RNA-Seq results between CC6 h and CC0 h (b)

下载: 全尺寸图片幻灯片

表 1 用qRT-PCR验证所用引物序列

Tab. 1 The sequence of primers used for qRT-PCR validation

基因名称	上游引物序列	下游引物序列
ADAMTS9	TGTGATGCTGGTGATTGCTTA	GCCTTCGTCTTCGGCTTTA
MMP16	TGGAAGTGGAATCGGTGGT	GGATGTAAAGCGAAGTTAGGGT
RARB	GCTCCAACCAGCACCTACCTT	AAACCCTTACACCCTTCGCA
SLC6A7	AAGTCATCGGGCAAGGTCG	GAAACAAGCAAAGCATCTCGGT
FCGBP	GAAGAACATCCTTGCCCCG	GACATCATACACGCAGTCATCATAG
PSAT1	GTAATGAGGGATTGGAAAAGCA	ACGCAACCCACCGACAGA

下载: 导出CSV

表 2 12个文库的基本信息

Tab. 2 The basic characteristic of reads in the 12 libraries

样本	原始测序数据	过滤后数据	过滤后碱基数/Gb	Q20值/%	Q30值/%	比对率/%
PC0 h-1	58 695 018	57 363 038	8.60	97.10	92.31	68.25
PC0 h-2	56 482 646	55 367 354	8.31	97.04	92.24	66.83
PC0 h-3	49 941 732	48 766 610	7.31	96.97	92.03	66.06
PC6 h-1	52 553 492	51 358 414	7.70	97.05	92.24	68.17
PC6 h-2	49 356 694	48 272 958	7.24	97.08	92.27	67.93
PC6 h-3	48 395 496	47 518 448	7.13	97.13	92.38	68.12
CC0 h-1	60 069 216	58 262 406	8.74	95.56	89.17	62.47
CC0 h-2	43 214 102	42 043 190	6.31	95.00	87.97	63.08
CC0 h-3	41 985 400	40 917 640	6.09	94.87	87.93	61.10
CC6 h-1	45 151 132	43 809 166	6.57	95.36	88.82	64.00
CC6 h-2	43 699 300	42 464 320	6.37	95.36	88.80	64.88
CC6 h-3	45 650 930	44 497 710	6.67	95.89	89.84	61.13
注：PC0 h代表吐脏前波里氏囊腔内体腔细胞；PC6 h代表吐脏后6 h波里氏囊腔内体腔细胞；CC0 h代表吐脏前体腔中体腔细胞；CC6 h代表吐脏后6 h体腔中体腔细胞。

下载: 导出CSV

表 3 显著富集通路中的差异表达基因

Tab. 3 The differential expression genes in significant enrichment pathways

基因名称	基因描述	表达倍数
Glycine，serine and threonine metabolism
PSAT1	phosphoserine aminotransferase isoform X1	1.71
GNMT	glycine N-methyltransferase	1.79
SARDH	Sarcosine dehydrogenase, mitochondrial	1.72
BHMT	betaine-homocysteine S-methyltransferase 1	1.48
ECM-receptor interaction
COL4A5	collagen alpha-5(IV) chain-like isoform X2	2.71
THBS-1	thrombospondin-1	2.49
COL9A2	collagen alpha-2(IX) chain	3.37
HSPG2	basement membrane-specific heparan sulfate proteoglycan core protein	2.58
COMP	cartilage oligomeric matrix protein	1.51
FoxO signaling pathway
PCK1	Phosphoenolpyruvate carboxykinase	2.40
GRB2	growth factor receptor-bound protein 2	1.54
NLK2	serine/threonine-protein kinase NLK2 isoform X1	1.91
Cyclin B	cyclin B	−2.06
PLK	polo-like kinase	−2.20
TGF-beta signaling pathway
FST	follistatin isoform X2	3.00

下载: 导出CSV

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