低温胁迫下军曹鱼幼鱼肝脏转录组测序分析

汤保贵; 蔡润佳; 张静

低温胁迫下军曹鱼幼鱼肝脏转录组测序分析

汤保贵^{1, 2,},
蔡润佳¹,
张静^{1, 2, ,}

1.
广东海洋大学水产学院，广东湛江，524088
2.
广东省水产经济动物病原生物学及流行病学重点实验室，广东湛江，524088

基金项目: 南方海洋科学与工程广东省实验室(湛江)项目(ZJW-2019-06)；湛江市海洋经济创新发展示范市项目(XM-202008-03)；国家海水鱼产业技术体系项目(CARS-47-G08)。

详细信息

作者简介:
汤保贵（1975— ），男，博士、副教授，主要研究方向为鱼类养殖与遗传育种。E-mail：zjtbg@163.com

通讯作者:
张静(1977— )，女，博士、副教授，主要研究方向为渔业生态环境与保护。E-mail：zjouzj@126.com

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- 文章访问数: 22
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出版历程
- 收稿日期: 2024-01-15
- 修回日期: 2024-11-11
- 网络出版日期: 2024-11-29

Transcriptome analysis of liver of juvenile cobia under low temperature stress

Tang Baogui^{1, 2
,},
Cai Runjia¹,
Zhang Jing^{1, 2
, ,}

1.
Fishery College, Guangdong Ocean University, Zhanjiang 524088, China
2.
Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088, China

摘要

摘要: 为研究低温胁迫对军曹鱼幼鱼的影响，将军曹鱼幼鱼饲养于常温（30.5±1.0℃）和低温（20.0±0.5℃）7 d后对军曹鱼肝脏进行有参转录组测序，每组3个生物学重复。结果显示，6个测序样品共检测到约243 694 134个raw reads，所有样品Q30%均超过94%，GC%在47.65%-48.16%范围内。共筛选出4 362个差异表达基因，其中2 793个基因上调，1 569个基因下调。KEGG通路富集分析结果显示，在脂质代谢中，大量差异基因富集在脂代谢过程、脂质生物合成过程、甘油磷脂代谢过程、磷脂代谢过程和甘油脂代谢过程等生物过程中， PPAR信号通路中PPARα、PPARβ、SCD-1、CPT-1和CPT-2等多个脂代谢相关基因对军曹鱼幼鱼应对低温胁迫具有关键作用；在糖代谢中，大量基因富集于糖酵解/糖异生、半乳糖代谢、淀粉和蔗糖代谢、戊糖和葡萄糖醛酸酯的相互转化等生物过程，其中G6PC、ENO等基因在军曹鱼幼鱼应对低温胁迫时发挥重要调节作用。
- 低温胁迫 /
- 军曹鱼 /
- 转录组 /
- 脂代谢 /
- 糖代谢
Abstract: In order to systematically study the effect of low temperature stress on the lipid metabolism of cobia juvenile, cobia juveniles were raised at normal temperature (30.5±1.0℃) and low temperature (20.0±0.5℃) for 7 days, and then the cobia livers were sequenced with genome-based transcriptome, and there were 3 biological replicates in each group. The research results show that a total of 243,694,134 row reads were found in 6 sequencing samples. The Q30% of all samples exceeded 94%, and the GC% was in the range of 47.65%-48.16%. A total of 4,362 differentially expressed genes were screened, of which 2,793 genes were up-regulated, and 1,569 genes were down-regulated. In terms of lipid metabolism, A large number of differential genes are enriched in biological processes such as lipid metabolism, lipid biosynthesis, glycerophospholipid metabolism, phospholipid metabolism and glycerideid metabolism, and also found that multiple lipid metabolism-related genes in the PPAR signaling pathway, such as PPARα, PPARβ, SCD-1, CPT-1, and CPT-2 play a key role in cobia juvenile under low temperature stress. In terms of glucose metabolism, a great many of genes are enriched in biological processes such as glycolysis/gluconeogenesis, galactose metabolism, starch and sucrose metabolism, and pentose and glucuronate interconversions. Notably, genes such as G6PC and ENO play crucial regulatory roles in the response of cobia juvenile to low temperature stress.
- low temperature stress /
- Rachycentron canadum /
- transcriptome /
- lipid metabolism /
- glucose metabolism

HTML全文

图 1 CG vs LG火山图

Fig. 1 CG vs LG volcano plot

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图 2 富集前20的GO terms

注：第一圈：富集前20的GOterm，圈外为基因数目的坐标尺，不同的颜色代表不同的Ontology；第二圈：背景基因中该GOterm的数目以及Q值，基因越多条形越长，Q值越小颜色越红；第三圈：上下调基因比例条形图，深紫色代表上调基因比例，浅紫色代表下调基因比例，下方显示具体的数值；第四圈：各GOterm的RichFactor值(该GOterm中差异基因数量除以所有数量)，背景网格线每一格代表0.1。

Fig. 2 The first 20 enriched GO terms

Note: The first circle: the first 20 enriched GO terms, outside the circle is the coordinate ruler of gene number, and different colors represent different Ontology; The second circle: the number and Q value of the GOterm in the background gene, the more genes, the longer the bars, and the smaller the Q value, the redder the color; The third circle: bar chart of the proportion of up and down genes, dark purple represents the proportion of up genes, light purple represents the proportion of down genes, and specific values are displayed below; The fourth circle: RichFactor value of each GOterm (the number of differential genes in the GOterm divided by all the numbers), and each grid line of the background represents 0.1.

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图 3 富集前20的pathways

Fig. 3 The first 20 enriched pathways

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图 4 PPAR信号通路图

注：红色框线表示：该框中的基因对比对照组，表达上调；绿色框线表示：该框中的基因对比对照组，表达下调；红-绿框线表示：该框中的基因对比对照组，部分表达上调，部分下调。

Fig. 4 PPAR signaling pathway

Note：The red box indicates that the genes in this box are up-regulated compared with the control group; the green box indicates that the genes in this box are down-regulated compared with the control group; the red-green box indicates that some genes in this box are up-regulated and some are down-regulated compared with the control group.

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图 5 转录组结果验证

Fig. 5 Verification of transcriptome results

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表 1 实时荧光定量PCR引物序列

Tab. 1 Primer sequences of genes used for RT-qPCR

基因名称 Gene name	基因编号 Gene ID	上游引物(5′-3′) Forward primer(5′-3′)	下游引物(5′-3′) Reverse primer(5′-3′)
ACC	Rca011875	TCGCCAGTCTCCCAACTCCTAT	ACCTGTCCACCTCCTCCTTCAT
FAS	Rca019519	AGCATCCTGTATCGCCCGTTTGA	GTCGGTCCTGTGGGTCTCCTTGT
HSL	Rca015361	AGCAGTCTGGTTTGGGTTTGGC	AGGTTCTGGGTAATGCGTTCA
CPT-1	Rca009591	TACCGCTTGGCTATGACTGGAC	TTGCTGGAGATGTGGAAGTTGATG
MGL	Rca013017	CACTGCGACCTTTGACCTCTTTG	AACCATCCTTCTGGGCGTAATC
PPARα	Rca010067	GAGTTCTCATCTTCCTCCTCATCGC	GGCACTTGTTGCGGTTCTTCTTT
β-actin	GU584189	AGGGAAATTGTGCGTGAC	AGGCAGCTCGTAGCTCTT
缩写：ACC，acetyl-CoA carboxylase，乙酰辅酶A羧化酶；FAS，fatty acid synthase，脂肪酸合成酶；HSL，hormone-sensitive triglyceride lipase，激素敏感性脂肪酶；CPT-1，carnitine palmityl transferase Ⅰ，肉碱脂酰转移酶Ⅰ；MGL，monoacylglycerol lipase，单酰甘油脂肪酶；PPARα，peroxisome proliferators-activated receptor，过氧化物酶体增殖物激活受体α。

下载: 导出CSV

表 2 样品过滤后测序质量评估统计表

Tab. 2 Statistical table of sequencing quality evaluation after sample filtering

处理	样品	RawDatas	CleanData(%)	CleanData(bp)	GC(%)	Q20(%)	Q30(%)
对照	GC-1	41052194	40443694 (98.52%)	6033805581	47.91%	98.36%	94.96%
	GC-2	39405242	38872552 (98.65%)	5802795648	47.66%	98.24%	94.66%
	GC-3	38277454	37694004 (98.48%)	5623819502	48.03%	98.30%	94.85%
低温	LG-1	41764832	41190146 (98.62%)	6148615460	47.65%	98.23%	94.74%
	LG-2	42025156	41532498 (98.83%)	6206078195	47.94%	98.19%	94.63%
	LG-3	41169256	40660388 (98.76%)	6075783071	48.16%	98.33%	94.90%

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表 3 测序结果比对基因组情况统计表

Tab. 3 Statistics table of sequencing results compared to the genome

处理	样品	Unmapped(%)	Unique_Mapped(%)	Multiple_Mapped(%)	Total_Mapped(%)
对照	GC-1	2505363(6.20%)	35435211(87.67%)	2477732(6.13%)	37912943(93.80%)
	GC-2	2677482(6.89%)	34013307(87.56%)	2154317(5.55%)	36167624(93.11%)
	GC-3	2784477(7.39%)	32773501(87.02%)	2103880(5.59%)	34877381(92.61%)
低温	LG-1	2918520(7.10%)	36014174(87.60%)	2177240(5.30%)	38191414(92.90%)
	LG-2	2690715(6.49%)	36329768(87.62%)	2440121(5.89%)	38769889(93.51%)
	LG-3	2159254(5.32%)	35571430(87.60%)	2875492(7.08%)	38446922(94.68%)

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表 4 脂代谢相关生物过程及通路的差异基因富集分析

Tab. 4 Differential gene enrichment analysis of lipid metabolism-related biological processes and pathways

数据库 database	功能/通路 Function/Access	基因数 genes number	编号 ID
GO	脂质代谢过程 lipid metabolic process	294	GO:0006629
	细胞脂质代谢过程 cellular lipid metabolic process	218	GO:0044255
	脂质生物合成过程 lipid biosynthetic process	82	GO:0008610
	细胞脂质分解代谢过程 cellular lipid catabolic process	26	GO:0044242
	脂质分解代谢过程 lipid catabolic process	30	GO:0016042
	中性脂质代谢过程 neutral lipid metabolic process	38	GO:0006638
	脂质稳态 lipid homeostasis	23	GO:0055088
	脂质定位 lipid localization	60	GO:0010876
	蛋白-脂质复合物亚基组织 protein-lipid complex subunit organization	11	GO:0071825
	脂质贮藏的积极调节 positive regulation of lipid storage	7	GO:0010884
	脂质储存的调节 regulation of lipid storage	14	GO:0010883
	脂质储存 lipid storage	15	GO:0019915
	脂质代谢过程的正向调节 positive regulation of lipid metabolic process	13	GO:0045834
	脂代谢过程调节 regulation of lipid metabolic process	55	GO:0019216
	甘油脂代谢过程 glycerolipid metabolic process	79	GO:0046486
	磷脂代谢过程 phospholipid metabolic process	82	GO:0006644
	脂质转运 lipid transport	48	GO:0006869
	脂质过氧化氢反应 response to lipid hydroperoxide	2	GO:0006982
KEGG	类固醇生物合成 Steroid biosynthesis	16	ko00100
	脂肪酸降解 Fatty acid degradation	25	ko00071
	类固醇激素的合成 Steroid hormone biosynthesis	24	ko00140
	初级胆汁酸生物合成 Primary bile acid biosynthesis	11	ko00120
	脂肪酸生物合成 Fatty acid biosynthesis	10	ko00061
	花生四烯酸代谢 Arachidonic acid metabolism	19	ko00590

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表 5 糖代谢相关生物过程及通路的差异基因富集分析

Tab. 5 Differential gene enrichment analysis of glucose metabolism-related biological processes and pathways

数据库 database	功能/通路 Function/Access	基因数 genes number	编号 ID
GO	单糖代谢过程 monosaccharide metabolic process	78	GO：0005996
GO	己糖代谢过程 hexose metabolic process	71	GO：0019318
KEGG	糖酵解/糖异生 Glycolysis/Gluconeogenesis	30	ko00010
	半乳糖代谢 Galactose metabolism	15	ko00052
	淀粉和蔗糖代谢 Starch and sucrose metabolism	16	ko00500
	戊糖和葡萄糖醛酸酯的相互转化 Pentose and glucuronate interconversions	17	ko00040

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