脂多糖对鲤肠上皮细胞转录组模式的调控分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0055

摘要/Abstract

摘要：

旨为探究脂多糖对鲤肠上皮细胞相关基因及功能的影响。本研究以鲤肠上皮细胞为试验对象,正常处理为对照组,脂多糖处理为实验组,对处理24 h的两组鲤肠上皮细胞进行转录组测序。转录组测序结果共获得44.77G高质量数据,其中,近81.83%的数据能够比对到鲤基因组。利用DESeq软件分析,与对照组相比,脂多糖处理可诱导产生差异表达基因（differentially expressed genes,DEG）590个,其中303个表达上调,287个表达下调。Gene Ontology（GO）功能富集分析发现,DEG在细胞过程、单有机体过程、代谢过程、细胞、细胞组分、细胞器、结合和催化活性功能亚类中所占比例最高。Kyoto Encyclopedia of Genes and Genomes（KEGG）通路途径分析发现,DEG显著富集于细胞周期、自噬、凋亡途径。并利用实时荧光定量PCR对随机挑选的10个差异基因进行验证,结果表明转录组数据可靠。该研究通过转录组测序技术,全面、快速地获取脂多糖暴露于鲤肠上皮细胞过程中的所有转录本信息,系统地证明脂多糖阻滞了鲤肠上皮细胞周期,诱导自噬,引起细胞凋亡,为脂多糖调控鲤肠上皮细胞分子机制提供了重要的理论依据。

关键词: 脂多糖, 鲤, 肠上皮细胞, 转录组测序, 实时荧光定量PCR

Abstract:

This work aims to explore the effect of lipopolysaccharide（LPS）on the genes and functions of intestinal epithelial cells in Cyprinus carpio. The intestinal epithelial cells of C. carpio were taken as the research object,normal treatment was as the control group,and lipopolysaccharide treatment was as the experimental group. Transcriptome sequencing was performed on the two groups of the intestinal epithelial cells of C. carpio treated for 24 h. A total 44.77G of high-quality data from transcriptome sequencing results were obtained,of which,nearly 81.83% data could be aligned to the C. carpio genome. Analyzed using the DESeq software and compared with the control group,590 differentially expressed genes（DEG）were induced by LPS treatment. Among them,303 were up-regulated and 287 were down-regulated. Gene Ontology（GO）functional enrichment analysis showed that DEG accounted for the highest proportion in cellular processes,single organism processes,metabolic processes,cells,cell part,organelle,binding and catalytic activity functional subclasses. The Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis revealed that DEG was significantly enriched in cell cycle,autophagy and apoptosis pathways. The 10 randomly selected differential genes were verified by quantitative real-time PCR,and the results showed that the transcriptome data were reliable. In this study,transcriptome sequencing technology was used to comprehensively and rapidly obtain all transcriptome information during LPS exposure to C. carpio intestinal epithelial cells,and to systematically prove that LPS blocked the cell cycle of C. carpio intestinal epithelial cells,induced autophagy and apoptosis,thus providing an important theoretical basis for the molecular mechanism of LPS regulation in C. carpio intestinal epithelial cells.

Key words: lipopolysaccharide, Cyprinus carpio, intestinal epithelial cells, transcriptome sequencing, quantitative real-time PCR

陈建军, 赵怡迪, 曹香林. 脂多糖对鲤肠上皮细胞转录组模式的调控分析[J]. 生物技术通报, 2021, 37(8): 213-220.

CHEN Jian-jun, ZHAO Yi-di, CAO Xiang-lin. Comprehensive Transcriptome Analysis of Intestinal Epithelial Cells of Cyprinus carpio Exposed to Lipopolysaccharide[J]. Biotechnology Bulletin, 2021, 37(8): 213-220.

图/表 7

参考文献 35

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Primer name	Gene bank	Oligonucleotide sequence（5'-3'）
β-actin	M24113.1	F：TCAGGACATTGAACCTCACTGTCT R：TAACACCATCACCAGAGTCCATCC
Ferritin	XM_019064890.1	F：ACGAGGACAGACTGAAGG R：AGCACAGATGGTCAACGA
LC3	XM_019072575.1	F：GCCATCCGACAGACCCTT R：TGAGCTGCAGTCTACGCC
Cathepsin	XM_019064744.1	F：ACCACACGCAACCAACAC R：CTCCACCATTGCAGGAAC
Casp9	XM_019066459.1	F：TGATCCCTCTGCCAGTCC R：TCTCCATCTTGTCGCAGT
Lamin	XM_019083381.1	F：TGGCTTTGGCTCAGGCTC R：GTCTCCGTCTCCAGCTGT
PARP	XM_019073641.1	F：GGTGCAGTCTCCCATGTT R：TCTGCTCTCCTTTCCCTC
BNIP3	XM_019071032.1	F：CATCAGTGCAGCCTCCTT R：TGTTCCGCTTTGGCTGTT
ULK1	XM_019089282.1	F：AGGGAAGCGGAAAGAGGA R：AATACCACAGCGAAGGCA
TP53INP2	XM_019070597.1	F：AGAGCTGAAGGGACCGAA R：ATGCTGGGGTGCTCGATG
LAMP	XM_019096275.1	F：CTGCATTGGCTGACATGG R：AGCTGGAAGGTGTTGAGA

Primer name	Gene bank	Oligonucleotide sequence（5'-3'）
β-actin	M24113.1	F：TCAGGACATTGAACCTCACTGTCT R：TAACACCATCACCAGAGTCCATCC
Ferritin	XM_019064890.1	F：ACGAGGACAGACTGAAGG R：AGCACAGATGGTCAACGA
LC3	XM_019072575.1	F：GCCATCCGACAGACCCTT R：TGAGCTGCAGTCTACGCC
Cathepsin	XM_019064744.1	F：ACCACACGCAACCAACAC R：CTCCACCATTGCAGGAAC
Casp9	XM_019066459.1	F：TGATCCCTCTGCCAGTCC R：TCTCCATCTTGTCGCAGT
Lamin	XM_019083381.1	F：TGGCTTTGGCTCAGGCTC R：GTCTCCGTCTCCAGCTGT
PARP	XM_019073641.1	F：GGTGCAGTCTCCCATGTT R：TCTGCTCTCCTTTCCCTC
BNIP3	XM_019071032.1	F：CATCAGTGCAGCCTCCTT R：TGTTCCGCTTTGGCTGTT
ULK1	XM_019089282.1	F：AGGGAAGCGGAAAGAGGA R：AATACCACAGCGAAGGCA
TP53INP2	XM_019070597.1	F：AGAGCTGAAGGGACCGAA R：ATGCTGGGGTGCTCGATG
LAMP	XM_019096275.1	F：CTGCATTGGCTGACATGG R：AGCTGGAAGGTGTTGAGA

Sample	CON_1	CON_2	CON_3	LPS_1	LPS_2	LPS_3
Total reads/M	52.55	54.38	50.14	50.75	54.24	51.36
Mapped reads/M	43.22	44.32	41.35	41.31	44.24	42.00
Mapped ratio/%	82.25	81.50	82.48	81.39	81.57	81.78
Clean bases/G	7.56	7.72	7.25	7.18	7.72	7.34
Q30/%	93.88	93.35	93.56	94.00	93.89	93.83
GC/%	46.91	46.27	46.45	46.50	47.62	47.38

Sample	CON_1	CON_2	CON_3	LPS_1	LPS_2	LPS_3
Total reads/M	52.55	54.38	50.14	50.75	54.24	51.36
Mapped reads/M	43.22	44.32	41.35	41.31	44.24	42.00
Mapped ratio/%	82.25	81.50	82.48	81.39	81.57	81.78
Clean bases/G	7.56	7.72	7.25	7.18	7.72	7.34
Q30/%	93.88	93.35	93.56	94.00	93.89	93.83
GC/%	46.91	46.27	46.45	46.50	47.62	47.38

	Gene name	Gene description	LPS -vs- CON Log₂（fold change）
自噬	LC3	微管相关蛋白轻链3	2.111361109
	BNIP3	B淋巴细胞瘤-2基因/腺病毒E1B相互作用蛋白3	3.959358016
	ULK1	自噬启动激酶ULK1	1.590396387
	TP53INP2	肿瘤蛋白p53诱导核蛋白2	3.655351829
	LAMP	溶酶体相关膜蛋白	3.987927168
	ATG16	自噬相关蛋白16	1.900464326
凋亡	Casp9	半胱氨酸天冬氨酸蛋白酶	3.774933444
	Cathepsin	组织蛋白酶	2.158941208
	Gadd45	生长阻滞和DNA损伤诱导蛋白45	2.436517227
	PARP	多聚ADP-核糖聚合酶	-3.684498174
	Lamin	核纤层蛋白	-4.006426269
	AP-1	激活蛋白1	2.264738712
细胞周期	PCNA	增殖细胞核抗原	-2.80580447
	Cdc14	细胞分裂周期蛋白	-1.920565533
	CycB	细胞周期调节蛋白B	-3.421463768
	CDK2	细胞周期蛋白依赖性激酶2	-2.038135129
	MCM2	微染色体维持蛋白2	-4.143929793
	PLK1	丝氨酸/苏氨酸蛋白激酶PLK1	-3.602036014
	TGF-β	转化生长因子-β	-2.509819643