基于转录组学筛选绵羊肉质性状相关候选基因

doi:10.7668/hbnxb.20192944

摘要/Abstract

摘要：

旨在分析滩羊与杜泊羊和小尾寒羊肉品质变化和肌肉内基因的表达情况,初步揭示它们肉质性状差异的分子机理,为其他绵羊品种选育与改良提供理论依据。通过对8月龄杜泊羊、滩羊和小尾寒羊的肉品质分析比较,并采用Illumina HiSeq^TM 4000平台对背最长肌组织进行转录组测序和分析,挖掘它们肉质性状差异的相关调控基因。结果显示:共筛选出820个差异表达基因,其中杜泊羊与滩羊之间99个,杜泊羊与小尾寒羊之间436个,滩羊与小尾寒羊之间552个。对差异基因进行GO功能富集分析结果显示,各比较组分别显著富集在224,517,657个GO条目中;KEGG通路富集分析表明,DEGs富集在cAMP、MAPK、AMPK、嘌呤代谢、甘油磷脂代谢、氨基酸和脂肪酸代谢以及糖酵解/异生等信号通路,进一步筛选出FOS、PLA2G4E、LPIN1、AMPD1、AMPD3、NT5C1A、GPI、PFKM和PKM等与肉品质调控和风味物质代谢有关的候选基因。对随机挑选的几个差异基因进行实时荧光定量PCR验证,结果显示,与转录组测序结果表达趋势一致,说明测序结果可靠,研究获得的这些差异基因可作为宁夏优质肉羊新品种(系)培育的基础资料。

关键词: 绵羊, 候选基因, 转录组, 肉品质, qRT-PCR

Abstract:

The aim of this study was to analyze the variation in meat quality and expression of intramuscular genes between Tan sheep,Dorper sheep and Small-tailed han sheep,and to preliminarily reveal the molecular mechanisms underlying the differences in their meat quality traits and also provide a theoretical basis for the selection and improvement of other sheep breeds. The meat quality of 8-month-old Dorper sheep,Tan sheep and Small-tailed han sheep was analyzed and compared,and the Illumina HiSeq^TM 4000 platform was used to sequence and analyze the transcriptome of the longest dorsal muscle tissue to explore the regulatory genes associated with the differences in their meat quality traits. The results showed that a total of 820 differentially expressed genes were screened,including 99 between Dorper sheep and Tan sheep,436 between Dorper sheep and Small-tailed han sheep and 552 between Tan sheep and Small-tailed sheep. The results of GO functional enrichment analysis of the differential genes showed that each comparison group was significantly enriched in 224,517 and 657 GO entries;and the KEGG pathway enrichment analysis showed that DEGs were enriched in cAMP,MAPK,AMPK,purine metabolism,glycerophospholipid metabolism,amino acid and fatty acid metabolism and glycolysis/gluconeogenesis signalling pathways,further screening for candidate genes related to meat quality regulation and flavor substance metabolism such as FOS,PLA2G4E,LPIN1,AMPD1,AMPD3, NT5C1A,GPI,PFKM and PKM. Real-time fluorescence quantitative PCR validation of several randomly selected differential genes showed consistent expression trends with the transcriptome sequencing results,indicating the reliability of the sequencing results. These differential genes obtained in this study can be used as basic information for the breeding of new breeds(lines)of Ningxia high-quality meat sheep.

Key words: Sheep, Candidate genes, Transcriptome, Meat quality, qRT-PCR

梁鹏, 张稳, 冯登侦, 强浩, 荣轩, 孟科. 基于转录组学筛选绵羊肉质性状相关候选基因[J]. 华北农学报, 2022, 37(4): 220-231. doi: 10.7668/hbnxb.20192944.

LIANG Peng, ZHANG Wen, FENG Dengzhen, QIANG Hao, RONG Xuan, MENG Ke. Screening Candidate Genes Related to Meat Quality Traits in Sheep Based on Transcriptome[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(4): 220-231. doi: 10.7668/hbnxb.20192944.

http://www.hbnxb.net/CN/Y2022/V37/I4/220

图/表 10

表1 引物信息

Tab.1 Primers information

基因 Gene
FOS	F: TGAAAACCATGACGGGAGGC	179
	R: TTGGTCTGTCTCCGCTTGGA
MEGF10	F: CTCAGGATCTCGGGTTGTTCT	168
	R: CTCTTGCACAGTCACGGAGT
GSTM3	F: GTGCCGCTTTGAGGCTTTG	160
	R: CCTACGGACGGAATGGAACA
NT5DC3	F: GAAGAGGAGCACGGAAGAACT	195
	R: TCGTTGATGAGAAGGTCCCG
PPARGC1A	F: TCTGTGTCACTGTGGATTGGA	117
	R: CAGAGCAGCACACTCGAT
UCP3	F: CTTTCCACTGGACACAGCCA	262
	R: GGTAACGATGCTGGAGTGGTC
CKM	F: CTCTGGAAGGGGACTTGCAT	283
	R: CATGATGAAGGGGTGACCTGG
RASD1	F: CGCAAGTTCTACTGCATCCG	119
	R: AGGATGAACACGTCTCCGGT
ACTC1	F: CTCACGGATTACCTCA	108
	R: AGCAACATAGCACAGC
AMPD1	F: CCAGCTGAAGGAAAACCTATTG	281
	R: TCGATGAGGGACAGTTTGGT
MSTN	F: ACAACTTTTGCCCAAGGCTC	154
	R: TGCTAGAAGATCAGACTCCGT
CCN1	F: GTACAGCAGCCTGAAAAAGGG	124
	R: GCAGGAACCGCAGTATTTGG
MYF6	F: ATAACTGCCAAGGAAGGAGGGA	114
	R: CACGCAGGGGAGTTTGTGTT
GAPDH	F: TGACGCTCCCATGTTTGTGA	162
	R: GCGTGGACAGTGGTCATAAGT

表2 绵羊肉品质差异分析

Tab.2 Analysis of sheep meat quality difference

指标 Indexes	杜泊羊 Dorper sheep	滩羊 Tan sheep	小尾寒羊 Small-tailed han sheep
肌纤维直径/μm Muscle fibre diameter	32.57±2.01a	27.38±1.34b	31.12±1.72ab
肌纤维密度/(根/mm²) Muscle fibre density	568.10±83.72b	921.58±58.99a	785.18±74.50a
剪切力/N Shear force	26.35±4.17b	21.42±2.02b	45.59±4.30a
失水率/% Water loss rate	35.73±1.30b	32.13±2.13b	42.39±1.41a
pH_24h	5.70±1.57b	5.37±0.08c	6.27±0.65a
脂肪/% Fat	3.41±0.41b	5.42±0.83a	2.10±0.31b
肌苷酸/(mg/g) Inosinic acid	1.40±0.22b	1.90±0.11a	2.08±0.12a
肌苷/(mg/g) Inosine	0.61±0.08a	0.37±0.03b	0.59±0.01a

表3 测序数据质量评估

Tab.3 Sequencing data quality assessment

样品 Sample	原始序列/条 Raw reads	有效序列/条 Clean reads	有效碱基/G Clean bases	错误率/% Error rate	Q20/%	Q30/%	GC含量/% GC content	比对数据/条 Mapped read	比对率/% Mapped ratio
D60	99 261 912	99 058 366	14.86	0.03	97.83	93.95	47.12	90 560 190	91.42
D70	82 978 304	82 579 838	12.39	0.03	97.84	93.92	49.94	73 906 060	89.50
D80	89 387 546	88 961 590	13.34	0.03	97.87	94.03	49.56	80 077 223	90.01
T50	89 605 464	89 190 166	13.38	0.02	98.25	94.85	48.99	80 837 548	90.64
T60	86 259 834	85 850 880	12.88	0.02	98.04	94.38	49.31	77 110 014	89.82
T70	88 543 880	88 113 542	13.22	0.02	98.01	94.31	49.33	79 352 964	90.06
XH5X	87 534 812	87 099 330	13.06	0.03	97.63	93.49	51.39	78 176 442	89.76
XH6X	95 229 800	94 732 306	14.21	0.03	97.85	94.07	52.04	84 738 074	89.45
XH7X	92 927 370	92 457 270	13.87	0.02	98.01	94.39	51.64	82 515 584	89.25

表4 各比较组合可变剪切分类和数量统计

Tab.4 Variable shear classification and quantity statistics for each comparison combination

类别 Category	D vs T		XHX vs T		XHX vs D
类别 Category	总数 Total	差异 Difference	总数 Total	差异 Difference	总数 Total	差异 Difference
外显子跳跃Skipped exon (SE)	15 967	135	16 135	151	16 884	181
外显子互斥Mutually exclusive exon (MXE)	2 061	19	1 932	16	2 132	19
5'端可变剪切Alternative 5' splice site (A5SS)	26	0	23	1	26	0
3'端可变剪切Alternative 3' splice site (A3SS)	153	1	135	2	146	3
内含子滞留Retained intron (RI)	32	1	33	0	34	0

图1 各样本表达水平分布每个区域的盒形图对应5个统计量(从上而下分别为最大值、上四分位数、中值、下四分位数和最小值)。

Fig.1 Distribution of expression levels by sample The box plot for each region corresponds to five statistics(maximum, upper quartile,median,lower quartile and minimum from top to bottom).

图2 DEGs层次聚类热图左侧根据表达相似程度对基因进行聚类,上方根据表达谱的相似程度对每个样本进行聚类,由蓝至红表达量逐渐上调,数字为均一化后的相对表达量。

Fig.2 Heat map of DEGs clustering The left side clusters the genes according to the degree of expression similarity,the top clusters each sample according to the degree of similarity of the expression profile,the expression is progressively up-regulated from blue to red,and the numbers are the relative expression after homogenization.

图3 DEGs的GO注释分类 A:1.细胞对镉离子的反应;2.细胞对铵离子的反应;3.含苯化合物代谢过程;4.骨吸收的正调控;5.骨重塑的正调控;6.中胚层细胞命运承诺;7.对镉离子的反应;8.硝基苯代谢过程;9.氮化合物解毒;10.含氮化合物的细胞解毒;11.毒素生物合成过程;12.生物碱代谢过程;13.二苯并对二噁英代谢过程;14.氧化脱乙基;15.组织重塑的正调控;16.单萜类代谢过程;17.翻译的昼夜调节;18.参与初级生殖层形成的细胞命运承诺;19.中胚层细胞分化;20.异源分解代谢过程;21.AP1复合体;22.核糖体小亚基;23.胞体纤维;24.血红蛋白复合物;25.生长细胞尖端;26.新生长细胞尖端;27.细胞尖端;28.精子纤维鞘;29.纤毛运动;30.嗜铬颗粒膜;31.曼切特;32.嗜铬颗粒;33.膜固有的;34.轴突丘;35.纤毛;36.核糖体亚单位;37.胞质小核糖体亚单位;38.膜神经元体细胞;39.氧化还原酶活性,作用于CH或CH2基团、醌或类似化合物作为受体;40.咖啡因氧化酶活性;41.芳香化酶活性;42.转铁蛋白受体活性;43.吡喃结构域结合;44.货物受体活性;45.犬尿氨酸3-单加氧酶活性;46. UDP-N-乙酰氨基葡萄糖-溶酶体-酶N-乙酰氨基葡萄糖磷酸转移酶活性;47.[硫酸乙酰肝素]-氨基葡萄糖3-磺基转移酶1活性;48. P物质受体活性;49.钙和钙调素反应性腺苷酸环化酶活性;50.RNA聚合酶Ⅱ调节区序列特异性DNA结合;51.RNA聚合酶Ⅱ调节区DNA结合;52.氧转运体活性;53.细胞骨架的结构成分;54.AMP脱氨酶活性;55.补体成分C1q结合;56.视蛋白结合;57.硫转移酶活性;58.速激肽受体活性。B:1.翻译;2.有机物生物合成过程;3.大分子生物合成过程;4.生物合成过程;5.细胞生物合成过程;6.细胞大分子生物合成过程;7.基因表达;8.细胞蛋白质代谢过程;9.蛋白质代谢过程;10.大分子代谢过程;11.代谢过程;12.蛋白质折叠;13.核糖体;14.核糖核蛋白复合物;15.胞浆核糖体;16.核糖体亚单位;17.胞浆大核糖体亚单位;18.肌原纤维;19.收缩纤维;20.大核糖体亚单位;21.非膜结合细胞器;22.细胞内非膜结合细胞器;23.肌节;24.胞浆部分;25.高分子络合物;26.收缩纤维部分;27.胞质小核糖体亚单位;28.多体核糖体;29.核糖体小亚基;30.条纹;31.核糖体的结构成分;32.结构分子活性;33.未折叠蛋白结合;34.rRNA结合。C:1.细胞趋化性;2.细胞对化学刺激的反应;3.蛋白质折叠;4.横纹肌细胞分化;5.肌细胞分化;6.呼吸爆发参与防御反应;7. 翻译;8.肌肉组织发育;9.肌肉结构发育;10.横纹肌组织发育;11.肌节;12.肌原纤维;13. 收缩纤维部分;14.收缩纤维;15.条纹;16.核糖体;17.胞浆核糖体;18.细胞外区;19.Z盘;20.胞浆部分;21.核糖体小亚基;22.未折叠蛋白结合;23.核糖体的结构成分;24.结构分子活性;25.rRNA结合;26.生长因子活性。图A代表D vs T组,图B代表XHX vs D组,图C代表XHX vs T组。图4,5同。

Fig.3 GO annotated classification of DEGs A:1.Cellular response to cadmium ion;2.Cellular response to ammonium ion;3.Benzene-containing compound metabolic process;4.Positive regulation of bone resorption;5.Positive regulation of bone remodeling;6.Mesodermal cell fate commitment;7.Response to cadmium ion;8.Nitrobenzene metabolic process;9.Detoxification of nitrogen compound;10.Cellular detoxification of nitrogen compound;11.Toxin biosynthetic process;12.Alkaloid metabolic process;13.Dibenzo-p-dioxin metabolic process;14.Oxidative deethylation;15.Positive regulation of tissue remodeling;16.Monoterpenoid metabolic process;17.Circadian regulation of translation;18.Cell fate commitment involved in formation of primary germ layer;19.Mesodermal cell differentiation;20.Xenobiotic catabolic process;21.AP1 complex;22.Small ribosomal subunit;23.Cell body fiber;24.Hemoglobin complex;25.Growing cell tip;26.New growing cell tip;27.Cell tip;28.Sperm fibrous sheath;29.Motile cilium;30.Chromaffin granule membrane;31.Manchette;32.Chromaffin granule;33.Intrinsic to membrane;34.Axon hillock;35.Cilium;36.Ribosomal subunit;37.Cytosolic small ribosomal subunit;38.Neuronal cell body membrane 39.Oxidoreductase activity,acting on CH or CH2 groups,quinone or similar compound as acceptor;40.Caffeine oxidase activity;41.Aromatase activity;42.Transferrin receptor activity;43.Pyrin domain binding;44.Cargo receptor activity;45.Kynurenine 3-monooxygenase activity;46.UDP-N-acetylglucosamine-lysosomal-enzyme N-acetylglucosamine phosphotransferase activity;47.[Heparan sulfate]-glucosamine 3-sulfotransferase 1 activity;48.Substance P receptor activity;49.Calcium-and calmodulin-responsive adenylate cyclase activity;50.RNA polymerase Ⅱ regulatory region sequence-specific DNA binding;51.RNA polymerase Ⅱ regulatory region DNA binding;52.Oxygen transporter activity;53.Structural constituent of cytoskeleton;54.AMP deaminase activity;55.Complement component C1q binding;56.Opsin binding;57.Sulfotransferase activity;58.Tachykinin receptor activity.B:1.Translation;2.Organic substance biosynthetic process;3.Macromolecule biosynthetic process;4.Biosynthetic process;5.Cellular biosynthetic process;6.Cellular macromolecule biosynthetic process;7.Gene expression;8.Cellular protein metabolic process;9.Protein metabolic process;10.Macromolecule metabolic process;11.Metabolic process;12.Protein folding;13.Ribosome;14.Ribonucleoprotein complex;15.Cytosolic ribosome;16.Ribosomal subunit;17.Cytosolic large ribosomal subunit;18.Myofibril;19.Contractile fiber;20.Large ribosomal subunit;21.Non-membrane-bounded organelle;22.Intracellular non-membrane-bounded organelle;23.Sarcomere;24.Cytosolic part;25.Macromolecular complex;26.Contractile fiber part;27.Cytosolic small ribosomal subunit;28.Polysomal ribosome;29.Small ribosomal subunit;30.I band;31.Structural constituent of ribosome;32.Structural molecule activity;33.Unfolded protein binding;34.rRNA binding.C:1.Cell chemotaxis;2.Cellular response to chemical stimulus;3.Protein folding;4.Striated muscle cell differentiation;5.Muscle cell differentiation;6.Respiratory burst involved in defense response;7.Translation;8.Muscle tissue development;9.Muscle structure development;10.Striated muscle tissue development;11.Sarcomere;12.Myofibril;13.Contractile fiber part;14.Contractile fiber;15.I band;16.Ribosome;17.Cytosolic ribosome;18.Extracellular region;19.Z disc;20.Cytosolic part;21.Small ribosomal subunit;22.Unfolded protein binding;23.Structural constituent of ribosome;24.Structural molecule activity;25.rRNA binding;26.Growth factor activity.Fig.A represents Group D vs T,Fig.B represents Group XHX vs D,Fig.C represents Group XHX vs T. The same as Fig.4,5.

图4 DEGs的KEGG富集分析 A:1.胆汁分泌;2.色氨酸代谢;3.昼夜夹带;4.卵巢类固醇生成;5.药物代谢-细胞色素P450;6.糖胺聚糖生物合成-硫酸乙酰肝素/肝素;7.安非他明成瘾;8.昼夜节律;9.cAMP信号通路;10.胰岛素分泌;11.亚油酸代谢;12.雌激素信号通路;13.可卡因成瘾;14.胆碱能突触;15.ABC转运;16.血管平滑肌收缩;17.长期抑郁症;18.细胞色素P450对外源性物质的代谢;19.多巴胺能突触;20.类固醇激素生物合成。B:1.核糖体;2.雌激素信号通路;3.昼夜夹带;4.胰岛素信号通路;5.2-氧羰基酸代谢;6.膀胱癌;7.抗原处理和呈递;8.缝隙连接;9.安非他明成瘾;10.谷氨酸能突触;11.淀粉和蔗糖代谢;12.军团菌病;13.慢性髓系白血病;14.酪氨酸代谢;15.p53信号通路;16.卵巢类固醇生成;17.酗酒;18.胰岛素分泌;19.ErbB信号通路;20.精氨酸和脯氨酸代谢。C:1.核糖体;2.粘着;3.军团菌病;4.雌激素信号通路;5.膀胱癌;6.胆汁分泌;7.肌动蛋白细胞骨架的调节;8.胃酸分泌;9.胰岛素信号通路;10.谷氨酸能突触;11.内质网中的蛋白质加工;12.精氨酸和脯氨酸代谢;13.安非他明成瘾;14.钙信号通路;15.前列腺癌;16.ErbB信号通路;17.甲状腺激素合成;18.疟疾;19.嘌呤代谢;20.血小板活化。

Fig.4 KEGG enrichment analysis of DEGs A:1.Bile secretion;2.Tryptophan metabolism;3.Circadian entrainment;4.Ovarian steroidogenesis;5.Drug metabolism-cytochrome P450;6.Glycosaminoglycan biosynthesis-heparan sulfate/heparin;7.Amphetamine addiction;8.Circadian rhythm;9.cAMP signaling pathway;10.Insulin secretion;11.Linoleic acid metabolism;12.Estrogen signaling pathway;13.Cocaine addiction;14.Cholinergic synapse;15.ABC transporters;16.Vascular smooth muscle contraction;17.Long-term depression;18.Metabolism of xenobiotics by cytochrome P450;19.Dopaminergic synapse;20.Steroid hormone biosynthesis.B:1.Ribosome;2.Estrogen signaling pathway;3.Circadian entrainment;4.Insulin signaling pathway;5.2-Oxocarboxylic acid metabolism;6.Bladder cancer;7.Antigen processing and presentation;8.Gap junction;9.Amphetamine addiction;10.Glutamatergic synapse;11.Starch and sucrose metabolism;12.Legionellosis;13.Chronic myeloid leukemia;14.Tyrosine metabolism;15.p53 signaling pathway;16.Ovarian steroidogenesis;17.Alcoholism;18.Insulin secretion;19.ErbB signaling pathway;20.Arginine and proline metabolism.C:1.Ribosome;2.Focal adhesion;3.Legionellosis;4.Estrogen signaling pathway;5.Bladder cancer;6.Bile secretion;7.Regulation of actin cytoskeleton;8.Gastric acid secretion;9.Insulin signaling pathway;10.Glutamatergic synapse;11.Protein processing in endoplasmic reticulum;12.Arginine and proline metabolism;13.Amphetamine addiction;14.Calcium signaling pathway;15.Prostate cancer;16.ErbB signaling pathway;17.Thyroid hormone synthesis;18. Malaria;19.Purine metabolism;20.Platelet activation.

表5 部分KEGG通路与DEGs富集结果

Tab.5 Results of partial enrichment of KEGG pathway with DEGs

KEGG通路名称KEGG pathway name	差异表达基因Differentially expressed gene
cAMP信号通路cAMP signaling pathway	FOS、CREM、ADCY8、ADCY1、GRIA3、DRD1、ATP1B4、ADCY10、ROCK2、PDE4C、CALM1、PDE4B、CREB3L3、CAMK2G
cGMP-PKG信号通路cGMP-PKG signaling pathway	CREM、ADCY8、ATP1B4、PLCB1、ADCY1、ROCK2、MYLK2、GUCY1A2、CALM1、CREB3L3、MYLK3、
TGF-β信号通路TGF-beta signaling pathway	SMAD1、MYC、THBS1、GDF6
MAPK信号通路MAPK signaling pathway	FOS、PLA2G4E、HSPB1、CACNG1、FLNC、MYC、HSPA1A、DUSP1、HSPA6、CACNA1I、GADD45G、FGF14、NFKB2、FLNC、DUSP10、MAP2K3
AMPK信号通路AMPK signaling pathway	CREM、PFKM、SLC2A4、PPARGC1A、GYS1、PFKFB2、CREB3L3、FBP2
PI3K-Akt信号通路PI3K-Akt signaling pathway	CREM、TNC、CDKN1A、HSP90AA1、THBS1、IL2RB、GYS1、MYC、PRLR、SPP1、COL11A2、COL11A1、MCL1、PDGFC、HSP90AB1、FGF14、RPS6、ITGB6、CDKN1A、CREB3L3、HSP90B1、LPAR1、FN1、CSF1
PPAR信号通路PPAR signaling pathway	FABP4、UBC、RXRG
嘌呤代谢Purine metabolism	PDE11A、AMPD3、ADCY8、AK4、ADCY1、NT5C1A、AMPD1、ADK、ADCY10、PKM、AK9、GUCY1A2、PDE4B、PDE4C、PAPSS2
甘油磷脂代谢Glycerophospholipid metabolism	PLA2G4E、DGKE、ETNPPL、CHAT、LPIN1、GPD2
氨基酸的生物合成Biosynthesis of amino acids	PFKM、CS、GOT1、IDH3A、PGAM1、PKM、GPT2
精氨酸和脯氨酸代谢Arginine and proline metabolism	MAOB、CKM、SMS、GOT1、ODC1、CKMT2、P4HA2、AMD1
肌动蛋白细胞骨架的调节Regulation of actin cytoskeleton	ENAH、DIAPH1、MRCL3、CYFIP2、MYL12B、ROCK2、SHC3、MYLK3、MYLK2、ITGB6、PARVA、FLNC、MRCL3、THBS1、TNC、MYL12B、PDGFC、COL1A1、CAPN2、SPP1、DIAPH1、FN1
脂肪细胞因子信号通路Adipocytokine signaling pathway	SOCS3、SLC2A4、PPARGC1A、RXRG
糖酵解/糖异生Glycolysis/Gluconeogenesis	PFKM、GPI、PGAM1、PKM、FBP2
脂肪酸代谢Fatty acid metabolism	ACOT7
肌醇磷酸代谢Inositol phosphate metabolism	PIK3C2G、PLCB1、PIP4K2B、ABCA1

表5 部分KEGG通路与DEGs富集结果

Tab.5 Results of partial enrichment of KEGG pathway with DEGs

KEGG通路名称KEGG pathway name	差异表达基因Differentially expressed gene
cAMP信号通路cAMP signaling pathway	FOS、CREM、ADCY8、ADCY1、GRIA3、DRD1、ATP1B4、ADCY10、ROCK2、PDE4C、CALM1、PDE4B、CREB3L3、CAMK2G
cGMP-PKG信号通路cGMP-PKG signaling pathway	CREM、ADCY8、ATP1B4、PLCB1、ADCY1、ROCK2、MYLK2、GUCY1A2、CALM1、CREB3L3、MYLK3、
TGF-β信号通路TGF-beta signaling pathway	SMAD1、MYC、THBS1、GDF6
MAPK信号通路MAPK signaling pathway	FOS、PLA2G4E、HSPB1、CACNG1、FLNC、MYC、HSPA1A、DUSP1、HSPA6、CACNA1I、GADD45G、FGF14、NFKB2、FLNC、DUSP10、MAP2K3
AMPK信号通路AMPK signaling pathway	CREM、PFKM、SLC2A4、PPARGC1A、GYS1、PFKFB2、CREB3L3、FBP2
PI3K-Akt信号通路PI3K-Akt signaling pathway	CREM、TNC、CDKN1A、HSP90AA1、THBS1、IL2RB、GYS1、MYC、PRLR、SPP1、COL11A2、COL11A1、MCL1、PDGFC、HSP90AB1、FGF14、RPS6、ITGB6、CDKN1A、CREB3L3、HSP90B1、LPAR1、FN1、CSF1
PPAR信号通路PPAR signaling pathway	FABP4、UBC、RXRG
嘌呤代谢Purine metabolism	PDE11A、AMPD3、ADCY8、AK4、ADCY1、NT5C1A、AMPD1、ADK、ADCY10、PKM、AK9、GUCY1A2、PDE4B、PDE4C、PAPSS2
甘油磷脂代谢Glycerophospholipid metabolism	PLA2G4E、DGKE、ETNPPL、CHAT、LPIN1、GPD2
氨基酸的生物合成Biosynthesis of amino acids	PFKM、CS、GOT1、IDH3A、PGAM1、PKM、GPT2
精氨酸和脯氨酸代谢Arginine and proline metabolism	MAOB、CKM、SMS、GOT1、ODC1、CKMT2、P4HA2、AMD1
肌动蛋白细胞骨架的调节Regulation of actin cytoskeleton	ENAH、DIAPH1、MRCL3、CYFIP2、MYL12B、ROCK2、SHC3、MYLK3、MYLK2、ITGB6、PARVA、FLNC、MRCL3、THBS1、TNC、MYL12B、PDGFC、COL1A1、CAPN2、SPP1、DIAPH1、FN1
脂肪细胞因子信号通路Adipocytokine signaling pathway	SOCS3、SLC2A4、PPARGC1A、RXRG
糖酵解/糖异生Glycolysis/Gluconeogenesis	PFKM、GPI、PGAM1、PKM、FBP2
脂肪酸代谢Fatty acid metabolism	ACOT7
肌醇磷酸代谢Inositol phosphate metabolism	PIK3C2G、PLCB1、PIP4K2B、ABCA1

图5 DEGs的qRT-PCR

Fig.5 qRT-PCR results for DEGs

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