Transcriptomics-based Screening of Candidate Genes for Intramuscular Fat Deposition in Jingyuan Chickens

doi:10.7668/hbnxb.20194927

Abstract

Abstract:

Intramuscular fat(IMF)is one of the important meat quality traits and an important indicator of chicken meat flavour.In order to investigate the mechanism of IMF formation at different developmental stages of Jingyuan chicken,the present study was carried out to screen candidate genes regulating IMF deposition in Jingyuan chicken by transcriptome sequencing using the pectoral muscles of 42(B1),126(B2),and 180(B3)day-old Jingyuan chicken(10 chickens at each stage).Real-time Fluorescence Quantitative PCR(RT-qPCR)was used to verify the reliability of the sequencing data.The results showed that 1 008 differentially expressed genes were identified in the B1vsB2 comparison group.1 396 differentially expressed genes were identified in the B1vsB3 comparison group. 696 differentially expressed genes were identified in the B2vsB3 comparison group.The GO function-enriched entries in the three comparison groups were mainly related to the positive regulation of transcription by RNA polymerase Ⅱ,cell adhesion,cell membrane and protoplasmic membrane,and binding of the same proteins.Meanwhile,PPAR signaling pathway,FOXO signaling pathway,insulin signaling pathway,MAPK signaling pathway,fatty acid degradation,and fatty acid metabolism were significantly enriched in KEGG pathway.Genes such as FABP1,FOXO1,FAS,and CPT1A,which were significantly enriched in these pathways,may be the key candidate genes to regulate IMF deposition in Jingyuan chicken.

Key words: Jingyuan chicken, Intramuscular fat, Transcriptome, Meat quality

CLC Number:

S831.2

HU Jiahuan, ZHAO Wei, YU Baojun, ZHAO Jinyan, FU Xi, ZHANG Tong, CHEN Yafei, GU Yaling, ZHANG Juan. Transcriptomics-based Screening of Candidate Genes for Intramuscular Fat Deposition in Jingyuan Chickens[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(S1): 329-339. doi: 10.7668/hbnxb.20194927.

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URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20194927

http://www.hbnxb.net/EN/Y2025/V40/IS1/329

Figures/Tables 12

Tab.1 Primer sequences of broilers

基因 Gene	基因登录号 Gene entry number	引物序列(5'-3') Primer sequences(5'-3')	退火温度/℃ Annealing temperature	产物长度/bp Product length
FAS	NM_001199487.2	F:TTGTTCGTCATCACCGTCTA	58	264
		R:TGCTTTGCTCAGTTTTGTCA
GLP2R	NM_001163248.1	F:ATGGAAGTGCAGGAAACGT	58	296
		R:GGCACGCAAGATGAAAGAT
ABCG1	XM_025145525.3	F:CGGTCCATCATCTGCACAAT	58	163
		R:GTACTGCCCTTACCAGTCGG
MSTN	NM_001001461.2	F:GAGACGATTATCACAATGCCTAC	58	294
		R:TTGCAGCACTGTCTTCACATC
PNPLA2	NM_001113291.2	F:AGCAGGGCTATCGGGATG	58	284
		R:TTGGGAGGCAAGTGTTCG
ACACB	XM_040648481.2	F:CTTCTCCCACCCAAATAGC	58	193
		R:CACTTCATCAAAGTTCCTCGT

Tab.2 IMF content of 42,126 and 180 days old Jingyuan chicken

个体号 Individual number	42日龄 42 days old	126日龄 126 days old	180日龄 180 days old
1	0.32	0.81	1.08
2	0.33	0.83	1.08
3	0.33	0.89	1.11
4	0.35	0.95	1.21
5	0.35	1.01	1.26
6	0.40	1.01	1.34
7	0.43	1.05	1.54
8	0.45	1.12	1.65
9	0.46	1.25	1.73
10	0.48	1.28	1.87
平均值±标准差	0.39±0.08	1.14±0.25	1.64±0.64
mean±s

Tab.3 Quality evaluation of sequencing data

样本名称 Sample name	原始读数 Raw reads	有效读数 Clean reads	有效读数/% Vaild reads	Q20/%	Q30/%	GC含量/% GC content
B1_1	44 641 996	43 603 376	97.67	99.95	96.95	49.50
B1_2	42 203 114	41 257 270	97.76	99.95	97.04	49.50
B1_3	44 595 972	43 532 480	97.62	99.95	96.98	49.00
B1_4	45 753 490	44 642 976	97.57	99.96	97.08	49.50
B1_5	49 088 696	47 828 862	97.43	99.95	96.92	49.00
B1_6	34 765 710	32 666 642	93.96	99.93	97.23	48.50
B1_7	34 482 536	32 364 054	93.86	99.94	97.22	48.50
B1_8	51 129 984	48 000 686	93.88	99.93	97.24	48.50
B1_9	36 737 614	34 327 564	93.44	99.93	97.20	48.50
B1_10	48 130 186	44 860 422	93.21	99.93	97.19	48.50
B2_1	36 313 250	35 568 816	97.95	99.96	97.40	47.50
B2_2	39 982 564	38 974 364	97.48	99.96	97.24	49.00
B2_3	40 405 820	39 409 366	97.53	99.96	97.42	48.50
B2_4	39 202 292	38 260 934	97.60	99.95	97.16	48.50
B2_5	39 477 124	38 527 366	97.59	99.96	97.24	49.50
B2_6	39 308 358	36 822 402	93.68	99.92	97.25	48.50
B2_7	54 702 700	51 272 360	93.73	99.92	97.13	48.50
B2_8	38 046 870	35 779 792	94.04	99.93	97.21	48.50
B2_9	37 486 270	35 152 722	93.77	99.93	97.17	48.50
B2_10	42 487 358	40 047 664	94.26	99.93	97.25	48.50
B3_1	39 399 168	38 287 554	97.18	99.96	97.29	49.50
B3_2	37 621 922	36 675 748	97.49	99.96	97.51	49.50
B3_3	47 136 518	46 038 676	97.67	99.95	96.80	49.50
B3_4	44 487 340	43 411 616	97.58	99.95	96.90	48.50
B3_5	45 429 302	44 357 812	97.64	99.96	96.96	49.00
B3_6	51 633 934	48 451 864	93.84	99.94	97.27	48.50
B3_7	52 871 666	49 369 848	93.38	99.93	97.07	48.50
B3_8	48 356 002	45 427 508	93.94	99.93	97.10	48.50
B3_9	39 153 982	36 827 204	94.06	99.93	96.99	48.00
B3_10	43 223 728	40 618 378	93.97	99.93	96.97	48.50

Fig.1 Volcano plot and cluster analysis of differentially expressed genes A,D.B1vsB2;B,E.B1vsB3;C,F.B2vsB3.Red.Up-regulated differentially expressed genes;Blue.Depressed differentially expressed genes;Grey.Genes with insignificant differences.

Fig.2 GO functional entries enriched for differentially expressed genes in the B1vsB2 comparison group 1.Positive regulation of transcription by RNA polymerase Ⅱ;2.G protein-coupled receptor signalling pathway;3.Positive regulation of transcription,DNA-templated;4.Signal transduction;5.Regulation of transcription by RNA polymerase Ⅱ;6.Regulation of transcription,DNA-templated;7.Immune response;8.Negative regulation of transcription by RNA polymerase Ⅱ;9.Protein phosphorylation;10.Positive regulation of gene expression;11.Inflammatory response;12.Cell adhesion;13.Ion transport;14.Negative regulation of transcription,DNA-templated;15.Chemokine-mediated signaling pathways;16.Positive regulation of ERK1 and ERK2 cascades;17.Positive regulation of apoptotic processes;18.Positive regulation of cell population proliferation;19.Negative regulation of apoptotic processes;20.Extracellular matrix organization;21.Negative regulation of cell population proliferation;22.Skeletal muscle tissue development;23.Chemotaxis;24.Positive regulation of protein kinase B signaling;25.T cell differentiation;26.Cytoplasm;27.Plasma membrane;28.Membrane;29.Nucleus;30.Integral component of membrane;31.Cytosol;32.Nucleoplasm;33.Extracellular space;34.Extracellular region;35.Mitochondrion;36.Integral component of the plasma membrane;37.Cytoskeleton;38.Endoplasmic reticulum;39.Golgi apparatus;40.Intracellular membrane-bounded organelle;41.Identical protein binding;42.RNA polymerase Ⅱ cis-regulatory regulartory region sequence-specific;43.DNA binding transcription factor activity;44.Metal ion binding;45.DNA binding;46.Sequence-specific double-stranded DNA binding;47.DNA binding transcription activator activity,RNA polymer;48.ATP binding;49.Sequence-specific DNA binding;50.G protein-coupled receptor activity.

Fig.3 GO functional entries enriched for differentially expressed genes in the B1vsB3 comparison group 1.Positive regulation of transcription by RNA polymerase Ⅱ;2.Regulation of transcription by RNA polymerase Ⅱ;3.Signal transduction;4.Extracellular matrix organisation;5.Cell adhesion;6.Negative regulation of cell population proliferation;7.G-protein-coupled receptor signaling pathway;8.Positive regulation of gene expression;9.Proteolysis;10.Positive regulation of cell population proliferation;11.Positive regulation of transcription,DNA-templated;12.Regulation of transcription,DNA-templated;13.Immune response;14.Negative regulation of transcription by RNA polymerase Ⅱ;15.Inflammatory response;16.Protein phosphorylation;17.Collagen fibre organisation;18.Cellular response to amino acid stimulus;19.Cell-cell adhesion;20.Negative regulation of transcription,DNA-templated;21.Negative regulation of cell migration;22.Positive regulation of cell migration;23.Cell differentiation;24.Angiogenesis;25.Cell migration;26.Membrane;27.Plasma membrane;28.Cytoplasm;29.Extracellular space;30.Integral component of membrane;31.Nucleus;32.Extracellular region;33.Cytosol;34.Nucleoplasm;35.Extracellular matrix;36.Integral component of plasma membrane;37.Cell surface;38.Basement membrane;39.Collagen-containing extracellular matrix;40.External side of plasma membrane;41.Identical protein binding;42.Metal ion binding;43.RNA polymerase Ⅱ cis-regulatory region sequence specific;44.DNA binding;45.DNA-binding transcription factor activity;46.Protein homodimerization activity;47.Hydrolase activity;48.ATP binding;49.Zinc ion binding;50.DNA-binding transcription factor activity,RNA polymerase.

Fig.4 GO functional entries enriched for differentially expressed genes in the B2vsB3 comparison group 1.Cell adhesion;2.Negative regulation of transcription by RNA polymerase Ⅱ;3.Collagen fibril organization;4.Extracellular matrix organization;5.Positive regulation of transcription,DNA-templated;6.Positive regulation of transcription by RNA polymerase Ⅱ;7.Regulation of transcription by RNA polymerase Ⅱ;8.Regulation of transcription,DNA-templated;9.Signal transduction;10.Cell migration;11.Positive regulation of gene expression;12.Lung development;13.Heart development;14.Negative regulation of cell population proliferation;15.Proteolysis;16.Positive regulation of cell-substrate adhesion;17.Cell response to amino acid stimulus;18.Blood vessel development;19.Positive regulation of apoptotic processes;20.Positive regulation of cell migration;21.Lipid metabolic processes;22.G protein-coupled receptor signaling pathway;23.Response to hypoxia;24.Bone mineralization;25.Regulation of membrane potential;26.Extracellular space;27.Membrane;28.Cytoplasm;29.Plasma membrane;30.Nucleus;31.Extracellular region;32.Integral component of the membrane;33.Extracellular matrix;34.Nucleoplasm;35.Cytosol;36.Collagen-containing extracellular matrix;37.Cell surface;38.Basement membrane;39.Integral component of plasma membrane;40.Intracellular membranes-bounded organelle;41.Identical protein binding;42.Metal ion binding;43.Calcium ion binding;44.Collagen binding;45.Heparin binding;46.RNA polymerase Ⅱ cis-regulatory region sequence-specific;47.DNA binding;48.Zinc ion binding;49.Protein homodimerization activity;50.Extracellular matrix structural constituent.

Fig.5 KEGG functional entries enriched for differentially expressed genes in the B1vsB2 comparison group 1.Cytokine-cytokine receptor interactions;2.Nitrogen metabolism;3.Glycine,serine and threonine metabolism;4.Arginine biosynthesis;5.Neuroactive ligand-receptor interaction;6.Alaine,aspartateand glutamate metabolism;7.Biosynthesis of amino acid;8.ECM-receptor interactions;9.Vascular smooth muscle contraction;10.Focal adhesion;11.Cysteine and methionine metabolism;12.TGF-beta signaling pathway;13.Adrenergic signaling in cardiomyocytes;14.Phagosome;15.Mucin type O-glycan biosynthesis biosynthesis;16.Ether lipid metabolism;17.VEGF signaling pathway;18.ErbB signaling pathway;19.Cell adhesion mplecules;20.Calcium signaling pathway.

Fig.6 KEGG functional entries enriched for differentially expressed genes in the B1vsB3 comparison group 1.ECM-receptor interaction;2.Focal adhesion;3.Cytokine-cytokine receptor interaction;4.Cell adhesion molecules;5.Arachidonic acid metabolism;6.Arginine biosynthesis;7.Hedgehog signaling pathway;8.Calcium signaling pathway;9.Alpha-linolenic acid metabolism;10.Linoleic acid metabolism;11.Cysteine and methionine metabolism;12.Phagosome;13.Neuroactive ligand-receptor interaction;14.Drug metabolism-cytochrome P450;15.Glycine,serine and threonine metabolism;16.Melanogenesis;17.Vascular smooth muscle contraction;18.Intestinal immune network for IgA production;19.Arginine and proline metabolism;20.Regulation of actin cytoskeleton.

Fig.7 KEGG functional entries enriched for differentially expressed genes in the B2vsB3 comparison group 1.ECM-receptor interaction;2.Focal adhesion;3.Adipocytokine signaling pathway;4.Arachidonic acid metabolism;5.Nitrogen metabolism;6.Fatty acid biosynthesis;7.Taurine and hypotaurinr metabolism;8.Linoleic acid metabolism;9.Hedgehog signalling pathway;10.Insulin signalling pathway;11.FOXO signalling pathway;12.Purine metabolism;13.Drug metabolism-other enzymes;14.RIG-l-like receptor signaling pathway;15.Apelin signaling pathway;16.Adrenergic signalling in cardiomyocytes;17.mTOR signaling pathway;18.Autophagy-animal;19.Phagosome;20.Calcium signaling pathway.

Tab.4 KEGG pathway associated with intramuscular fat deposition with differentially expressed mRNA enrichment results

比较组 Comparison group	KEGG富集通路 KEGG enrichment pathway	基因名称 Gene name	差异倍数 Log₂ (Fold Change)	比较组 Comparison group	KEGG富集通路 KEGG enrichment pathway	基因名称 Gene name	差异倍数 Log₂ (Fold Change)
B1vsB2	MAPK 信号通路	TGFB3	1.95	B2vsB3	FOXO 信号通路	IRS4	1.03
		DUSP1	1.26			TGFB3	-2.39
	FOXO 信号通路	TGFB3	1.95			FOXO1	1.05
		FOXO1	-1.40		脂肪细胞因子信号通路	IRS4	1.03
	PPAR 信号通路	CPT1A	-1.55			CPT1A	1.76
		FABP1	1.19			ACACB	1.68
	胰岛素信号通路	FOXO1	-1.40		胰岛素信号通路	IRS4	1.03
	脂肪酸降解	CPT1A	-1.55			FOXO1	1.05
	脂肪酸代谢	CPT1A	-1.55			ACACB	1.68
	脂肪细胞因子信号通路	CPT1A	-1.55		脂肪酸降解	CPT1A	1.76
B1vsB3	代谢途径	PNPLA2	1.82		PPAR 信号通路	CPT1A	1.76
	甘油酯代谢	PNPLA2	1.82		脂肪酸代谢	ACACB	1.68
	脂肪酸的生物合成	ACACB	1.54			CPT1A	1.76
	胰岛素信号通路	ACACB	1.54		脂肪酸的生物合成	ACACB	1.68
	丙酮酸代谢	ACACB	1.54		代谢途径	PNPLA2	1.10
	脂肪细胞因子信号通路	ACACB	1.54			ACACB	1.68
	MAPK 信号通路	FAS	-1.27		丙酮酸代谢	ACACB	1.68
	ABC 转运体	ABCG1	-2.96			CPT1A	1.76
B2vsB3	MAPK 信号通路	TGFB3	-2.39		甘油酯代谢	PNPLA2	1.10

Fig.8 Comparative RT-qPCR analysis of differentially expressed genes Different lowercase letters indicate significant differences between different comparisons(P<0.05).

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