生物技术通报 ›› 2021, Vol. 37 ›› Issue (5): 56-66.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0919
收稿日期:
2020-07-22
出版日期:
2021-05-26
发布日期:
2021-06-11
作者简介:
邱小宇,男,硕士研究生,研究方向:动物肉品质调控;E-mail: 基金资助:
QIU Xiao-yu1(), LIU Zuo-hua1,2, QI Ren-li1,3()
Received:
2020-07-22
Published:
2021-05-26
Online:
2021-06-11
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。
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
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%) |
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
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 |
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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