基于转录组测序和血清指标测定对牛肾周脂肪的脂质代谢和内分泌功能解析

doi:10.7668/hbnxb.20193065

摘要/Abstract

摘要：

旨在筛选西门塔尔牛与安格斯牛肾周脂肪的差异表达基因,分析2个品种牛血清指标差异,以期对肾周脂肪的脂质代谢和内分泌功能进行解析,为牛品种选育打下基础。选取相同月龄的西门塔尔牛和安格斯牛各3头,饲喂前采集空腹血液分离血清进行血清生化指标测定;屠宰后,采取相同大小的肾周脂肪,提取总RNA后进行转录组测序分析,使用DEGseq方法对各个样品的基因表达水平进行差异检测,对差异表达基因进行GO分析和KEGG分析,并使用实时荧光定量PCR验证测序结果。结果表明,安格斯牛血清中白蛋白(ALB2)、尿素氮(UREAL)和葡萄糖(GLUC3)含量显著低于西门塔尔牛(P<0.05),甘油三酯(TRIGL)含量显著高于西门塔尔牛(P<0.05)。转录组结果显示,2个品种牛肾周脂肪间的差异表达基因为1 743,其中1 361个基因上调表达,382个基因下调表达。差异表达基因GO功能富集结果显示,差异基因显著富集到52个生物学功能,主要与细胞过程和细胞有关;KEGG富集结果显示,差异表达基因主要在Fat digestion and absorption、cAMP、PPAR和Rap1等信号通路显著富集,最终筛选出与脂质代谢相关的基因SCD5、APOA4、PTX3、OLR1和PRKCZ。通过实时荧光定量PCR测定差异基因的相对表达量,结果显示,测序结果和实时荧光定量PCR结果一致,说明测序结果可靠,研究获得的差异基因可作为肉牛品种培育的基础资料。

关键词: 牛, 肾周脂肪, 脂质代谢, 血清生化指标, 转录组

Abstract:

This study aims to screen the differentially expressed genes in perirenal fat of Simmental cattle and Angus cattle,and analyze the differences in serum indexes between the two breeds,in order to determine the lipid metabolism and endocrine function of perirenal fat,so as to lay a foundation for cattle breed selection.Three Simmental cattle and three Angus cattle of the same age were selected,and fasting blood was collected before feeding to separate and for determination of serum biochemical indexes.After slaughter,perirenal fat of the same size was sampled,and total RNA was extracted for transcriptome sequencing analysis.The differentially expressed genes(DEGs)were identified using the DEGseq method,and GO and KEGG analysis were performed.The sequencing results were verified using Real-time fluorescence quantitative PCR.The results showed that:the serum contents of albumin(ALB2),urea nitrogen(UREAL)and glucose(GLUC3)in Angus cattle were significantly lower than that of Simmental cattle(P<0.05),and the contents of triglyceride(TRIGL)were significantly higher than that of Simmental cattle(P<0.05).Transcriptome results showed that 1 743 genes were differentially expressed between the perirenal fats of two breeds.Among that,1 361 and 382 genes were up regulated and down regulated,respectively.GO analysis showed that DEGs were significantly enriched to 52 biological functions,which were mainly related to cellular processes and cells.KEGG analysis showed that DEGs were significantly enriched mainly in fat digestion and absorption,cAMP,PPAR and Rap1 signaling pathways.Five genes(SCD5,APOA4,PTX3,OLR1 and PRKCZ)related to lipid metabolism were identified,and the sequencing results of these five genes were verified by Real-time fluorescence quantitative PCR,these results indicated that the sequencing results were reliable and the obtained differential genes could be used as basic data for breeding beef cattle breeds.

Key words: Cattle, Perirenal fat, Lipidmetabolism, Serum biochemical indexes, Transcriptome

王利香, 刘迪, 尤欢, 王云龙, 于万超, 高树新. 基于转录组测序和血清指标测定对牛肾周脂肪的脂质代谢和内分泌功能解析[J]. 华北农学报, 2023, 38(1): 210-217. doi: 10.7668/hbnxb.20193065.

WANG Lixiang, LIU Di, YOU Huan, WANG Yunlong, YU Wanchao, GAO Shuxin. Analysis of Lipid Metabolism and Endocrine Function of Perirenal Fat in Cattle Based on Transcriptome Sequencing and Serum Index Measurements[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 210-217. doi: 10.7668/hbnxb.20193065.

http://www.hbnxb.net/CN/Y2023/V38/I1/210

图/表 9

表1 引物信息

Tab.1 Primer information

基因 Gene	序列(5'—3') Sequence(5'—3')	NCBI登录号 Accession number	扩增长度/bp Amplification size
APOA4	F:CAAGAAGGCGGTGGAACAT	NM_001037480.1	102
	R:AGGTCGTCCGTGTAGGTGC
PTX3	F:CTATCGGTCCATAATGCTTGT	NM_001076259.2	111
	R:TTCTTTGAATCCCAGGTGC
OLR1	F:CTTTGTCTGGGATTACTGG	NM_174132.2	196
	R:CAAGGGTTTCTATCATTTCT
PRKCZ	F:CCCCGACATGAACACTGAA	NM_001077833.2	184
	R:GAAGAAGGCGTGAGACTTGA
SCD5	F:TGGCTGGTGAACAGTGTCG	NM_001076945.1	147
	R:CGCAGAGTAGTCAAAGGGAAA
β-actin	F:TCCTGGGTATGGAGTCCTG	NM_001033618.1	186
	R:TGATCTTCATCGTGCTTGG

表2 血清生化指标结果

Tab.2 Results of blood physiological and biochemical indexes

指标 Index	西门塔尔牛 Simmental cattle	安格斯牛 Angus cattle	P值 P-value
ALTL/(U/L)	37.61±8.20	39.32±6.08	0.265
ASTL/(U/L)	84.88±22.49	87.10±23.31	0.646
TP2/(g/L)	75.53±4.51	74.18±4.11	0.141
ALB2/(g/L)	39.39±3.34	36.69±1.81	0.036
TRIGL/(mmol/L)	0.24±0.08	0.34±0.11	0.017
GLUC3/(mmol/L)	3.34±0.21	2.94±0.37	0.041
UREAL/(mmol/L)	5.45±0.82	4.32±0.50	0.033
CHOL2/(mmol/L)	2.26±0.37	2.33±0.52	0.479
HDLC4/(mmol/L)	2.53±0.47	2.53±0.51	0.995
PHOS2/(mmol/L)	2.43±0.28	2.40±0.26	0.700
CA2/(mmol/L)	2.42±0.19	2.13±0.16	0.095

表3 过滤后的reads质量

Tab.3 Clean reads quality

样品 Sample	总原始reads/Mb Total raw reads	过滤后reads/Mb Clean reads	过滤后reads Q20/% Clean reads Q20	过滤后reads Q30/% Clean reads Q30	过滤后reads 占比/% Clean reads ratio
An_PF_1	127.00	110.84	96.81	89.19	87.27
An_PF_2	124.47	110.14	96.87	89.18	88.49
An_PF_3	126.95	109.34	96.48	88.28	86.13
An_PF_ $X ¯$	126.14	110.11	96.72	88.88	87.30
Si_PF_1	124.44	109.22	96.72	88.60	87.77
Si_PF_2	127.00	111.21	96.73	88.84	87.57
Si_PF_3	127.00	110.42	96.69	88.84	86.94
Si_PF_ $X ¯$	126.15	110.28	96.71	88.76	87.43

表3 过滤后的reads质量

Tab.3 Clean reads quality

样品 Sample	总原始reads/Mb Total raw reads	过滤后reads/Mb Clean reads	过滤后reads Q20/% Clean reads Q20	过滤后reads Q30/% Clean reads Q30	过滤后reads 占比/% Clean reads ratio
An_PF_1	127.00	110.84	96.81	89.19	87.27
An_PF_2	124.47	110.14	96.87	89.18	88.49
An_PF_3	126.95	109.34	96.48	88.28	86.13
An_PF_ $X ¯$	126.14	110.11	96.72	88.88	87.30
Si_PF_1	124.44	109.22	96.72	88.60	87.77
Si_PF_2	127.00	111.21	96.73	88.84	87.57
Si_PF_3	127.00	110.42	96.69	88.84	86.94
Si_PF_ $X ¯$	126.15	110.28	96.71	88.76	87.43

图1 差异基因火山图

Fig.1 Differential gene volcano map

图2 西门塔尔牛和安格斯牛肾周脂肪的差异表达基因GO功能分类

Fig.2 Go function classification of differentially expressed genes in perirenal fat of Simmental and Angus cattle

图3 西门塔尔牛和安格斯牛肾周脂肪的差异表达基因Pathway分类图

Fig.3 Pathway classification of differentially expressed genes in perirenal fat of Simmental and Angus cattle

表4 差异表达基因KEGG富集分析

Tab.4 Enrichment analysis of differentially expressed gene KEGG

信号通路 Signaling pathway	通路ID Pathway ID	差异基因数量 Number of DEGs	Q值 Q-value
脂肪消化与吸收信号通路 Fat digestion and absorption signaling pathway	ko04975	36	0.008
cAMP信号通路 cAMP signaling pathway	ko04024	65	0.000
PPAR信号通路 PPAR signaling pathway	ko03320	35	0.008
Rap1信号通路 Rap1 signaling pathway	ko04015	55	0.005

表5 差异表达基因DEG检测结果

Tab.5 Detection results of differentially expressed gene DEG

基因ID Gene ID	基因名称 Gene name	基因注释 Gene annotation	差异表达倍数 log₂Ratio
537301	APOA4	载脂蛋白A4	1.44
541148	PTX3	正五聚蛋白3	3.50
281368	OLR1	氧化低密度脂蛋白受体1	1.99
286877	PRKCZ	蛋白激酶C	3.73
617419	SCD5	硬脂酰辅酶A去饱和酶5	1.25

图4 西门塔尔牛安格斯牛肾周脂肪差异基因qPCR与RNA-seq的表达量

Fig.4 Expression of perinephric fat differential gene qPCRand RNA-seq in Simmental and Angus cattle

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