无菌猪和普通猪早期脂肪发育及脂肪组织基因转录表达的差异

doi:10.13560/j.cnki.biotech.bull.1985.2020-0919

摘要/Abstract

摘要：

通过比较无菌仔猪和普通仔猪在生长早期脂肪沉积的差异及脂肪组织基因转录表达谱的不同,评估肠道微生物定植对猪脂肪组织早期发育的直接影响。分别采集25日龄无菌仔猪（3头）和普通仔猪（3头）的颈部皮下脂肪,切片后经HE染色观察不同猪脂肪细胞的形态差异;Western Blot检测脂肪合成调控因子的蛋白表达差异;ELISA法测定不同猪脂肪组织分泌产生的脂肪细胞因子含量;转录组测序（RNA-seq）分析不同猪脂肪组织中的基因表达谱,鉴定关键差异基因及其相关信号网络。与普通仔猪相比,相同日龄的无菌仔猪体脂沉积量较少,皮下脂肪厚度和脂肪细胞尺寸均明显减小（P<0.001）;脂肪组织中的脂肪酸结合蛋白4（fatty acid binding protein 4,FABP4）、过氧化物酶体增殖体激活受体γ（peroxisome proliferators activated recepor γ,PPARγ）、乙酰辅酶A羧化酶（acetyl CoA carboxylase,ACC）、脂肪酸合成酶（fatty acid synthase,FAS）的蛋白表达水平显著降低（P<0.05）。脂肪产生的功能性细胞因子脂联素（adiponectin,P=0.100）和瘦素（leptin,P=0.095）有着不同程度的减少。测序结果显示与普通猪相比,无菌猪的脂肪中有695个基因显著差异表达（P<0.05）,包括338个基因表达上调,357个基因表达下调。qRT-PCR验证了转录组测序结果的准确性。KEGG通路富集分析结果显示差异基因主要与脂质合成代谢、分解代谢、脂质氧化、能量稳态相关。肠道微生物的定植显著影响了动物脂肪组织的发育、代谢和生理功能。

关键词: 肠道微生物, 脂肪, 基因, 转录组, 猪

Abstract:

We compared the difference of fat deposition in the early growth stage and the different transcriptional profiles of adipose tissue in sterile piglets and normal piglets,and evaluated the direct effects of intestinal microbial colonization on the early development of pig adipose tissue. The cervical subcutaneous fat of 25-day sterile piglets（3 in GF piglet group）and normal pigs（3 in normal piglet group）was collected. HE staining was used to observe the morphological differences of fat cells,Western Blot to detect the protein expression differences of fat synthesis regulators,ELISA to detect the content of cytokines secreted in fat tissues,and RNA-seq to analyze the gene expression profile differences in fat tissues and identify the key differential genes and relevant signal networks. Compared with the normal piglets,the fat deposition in the same age of the sterile piglets was less,and the thickness of back fat and the sizes of fat cells significantly reduced（P<0.01）. The expression of fatty acid binding protein 4（FABP4）,peroxisome proliferators activated receptor γ（PPARγ）,acetyl CoA carboxylase（ACC）and fatty acid synthase（FAS）in the fat decreased significantly（P<0.05）. Adiponectin（P=0.100）and leptin（P=0.095）also decreased in varied degrees. The results of transcriptome sequencing showed that there were 695 genes in the fat of sterile piglets,including 338 genes up-regulated and 357 genes down-regulated（P<0.05,fold change>2）. Accuracy of RNA-sequencing data was verified by the qRT-PCR results. KEGG analysis showed that the differentially expressed genes were mainly related to lipid synthesis metabolism,catabolism,lipid oxidation and energy homeostasis. The colonization and status of intestinal microorganisms significantly affect the development,metabolism and function of animal adipose tissue.

Key words: gut microorganism, fat, gene, transcriptome, pig

邱小宇, 刘作华, 齐仁立. 无菌猪和普通猪早期脂肪发育及脂肪组织基因转录表达的差异[J]. 生物技术通报, 2021, 37(5): 56-66.

QIU Xiao-yu, LIU Zuo-hua, QI Ren-li. Differences in Early Fat Development and Gene Transcription Expression in the Adipose Tissues of Piglets with and Without Gut Microbiota[J]. Biotechnology Bulletin, 2021, 37(5): 56-66.

图/表 9

图1 无菌仔猪和普通仔猪脂肪沉积的差异 A：无菌仔猪和普通仔猪脂肪组织HE染色;B：无菌仔猪和普通仔猪颈部皮下脂肪厚度;C：无菌仔猪和普通仔猪脂肪组织中脂肪细胞直径。*** P<0.001;**** P<0.0001

Fig. 1 Difference of fat deposition between GF pigs and normal pigs A: He staining of fat tissue of GF pigs and normal pigs. B: Back-fat of GF pigs and Normal pigs. C: Diameter of fat cells in fat tissue of GF pigs and Normal pigs. *** P<0.001;**** P<0.0001

图2 无菌猪和普通猪的脂肪合成调控分子表达和脂肪细胞因子含量的差异 A：无菌仔猪和普通仔猪脂肪组织FABP4、PPARγ、ACC、FAS的差异表达;B：无菌仔猪和普通仔猪脂肪组中的脂联素（Adiponection）的含量;C：无菌仔猪和普通仔猪脂肪组织瘦素（Leptin）的含量。*P<0.05;**P<0.01

Fig.2 Differences in fat synthesis regulatory factors and adipocytokines between GF pigs and normal pigs A: The differential expression of FABP4, PPAR γ, ACC and FAS in adipose tissue of GF and Normal piglets. B: The differential secretion of adiponectin. C: The differential secretion of adiponectin leptin. * P<0.05;** P<0.01

表1 测序数据基因组比对统计

Table 1 Statistical list of mapping to genome

样品名 Sample name	总序列数 Raw reads	干净序列数 Clean reads	总对比到的序列数 Total mapped reads	多重对比的序列数 Multiple mapped reads	唯一对比的序列数 Uniquely mapped reads
Normal A1	60 328 616	59 694 488	50 892 462（85.25%）	4 145 489（6.94%）	46 746 973（78.31%）
Normal A2	53 878 956	53 400 340	45 855 826（85.87%）	4 209 183（7.88%）	41 646 643（77.99%）
Normal A3	65 393 020	64 761 370	55 361 124（85.48%）	5 082 167（7.85%）	50 278 957（77.64%）
GF A1	48 721 914	48 260 760	41 315 919（85.61%）	2 945 880（6.10%）	38 370 039（79.51%）
GF A2	54 262 278	53 749 144	46 093 576（85.76%）	3 467 323（6.45%）	42 626 253（79.31%）
GF A3	57 431 858	56 874 014	48 683 278（85.60%）	3 484 909（6.13%）	45 198 369（79.47%）

图3 样本测序基因覆盖度

Fig.3 Coverage of sequencing

图4 样本基因表达分布和相关性 A：基因表达量分布情况;B：样本相关性

Fig.4 Distribution and correlation of genes expression patterns in different samples A：The distribution of gene expression. B：Correlation of samples

图5 差异表达基因 A：差异表达基因维恩图;B：差异基因火山图;C：差异基因聚类图

Fig.5 Differentially expressed genes A：The Veen map of differentially expressed gene. B：The volcano map of differentially expressed gene. C：The clustering map of differentially expressed gene

表2 十个表达差异最显著的上调和下调基因

Table 2 Top 10 up or down expressed genes

基因名称Gene Name	log2FC	变化Change	功能Function
TNS4	4.756	Up	Function unknown
TMP	4.587	Up	Function unknown
NRIP3	4.305	Up	Proteolysis,aspartic-type endopeptidase activity
MAPKAPK	4.088	Up	ATP binding,protein kinase activity
PRG4	4.076	Up	Receptor-mediated endocytosis
IRF5	4.051	Up	Function unknown
IRF6	4.007	Up	Keratinocyte proliferation
ITIH3	3.668	Up	Hyaluronan metabolic process
3,5,3’,5’- tetraiodothyronine	3.566	Up	Hormone biosynthetic process
Phenylalanine 4-monooxygenase	3.55	Up	Amino acid transport and metabolism,oxidoreductase activity
C4BPA	-7.537	Down	Positive regulation of protein catabolic
SQRDL	-6.269	Down	Oxidoreductase activity
PIK3C2G	-4.847	Down	Phosphatidylinositol 3-kinase（PI3K）complex
KCNIP2	-4.293	Down	Cell cycle control,myosin light chain
LIPG	-4.149	Down	Lipid metabolic,high-density lipoprotein particle remodeling
MAP6	-3.894	Down	Lysosome localization
CES1	-3.625	Down	Lipid transport and metabolism,Carboxylesterase
CYP1A1	-3.588	Down	Steroid metabolic,Steroid hormone biosynthesis
SucCβ	-3.498	Down	Fatty acid biosynthetic process,acetyl-CoA biosynthetic process
LRFN2	-3.452	Down	Function unknown

图6 差异表达基因的qRT-PCR验证 A：转录组测序结果;B：qRT-PCR结果

Fig.6 Verification of the differentially expressed genes by qRT-PCR A：RNA-sequencing data. B：qRT-PCR data

图7 差异表达基因的KEGG功能富集 A：下调基因的KEGG分析;B：上调基因的KEGG分析

Fig.7 KEGG enrichment analysis of the differentially expressed genes A： KEGG analysis of the down-regulated genes. B：KEGG analysis of the up-regulated genes

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