基于肝脏转录组测序筛选康乐黄鸡开产相关基因和关键通路

doi:10.7668/hbnxb.20194898

摘要/Abstract

摘要：

通过分析开产与未开产康乐黄鸡肝脏组织转录组表达谱,筛选出开产相关候选基因和关键通路,为研究鸡肝脏基因调控开产性状分子机理提供理论基础,为康乐黄鸡选种选育提供一定参考。选取154日龄已开产(H组)、未开产(L组)各3个个体的肝脏组织,采用TRIzol法提取总RNA,利用Illumina测序平台进行转录组测序,筛选2组个体的差异表达基因;对差异表达基因进行功能富集和蛋白互作分析;随机选取9个候选基因,在9个个体肝脏中,进行qRT-PCR验证。一共检测到21 465个基因在肝脏组织表达,共筛选出227个差异表达基因,其中48个表达上调,179个表达下调。qRT-PCR验证结果表明,9个基因在2组个体肝脏中表达量变化趋势与RNA-Seq结果基本一致,且在H、M(即将开产)和L 3组个体中呈现表达量依次递减或递增的趋势。初步确定了6个开产性状相关候选基因,分别是VTG1、VTG2、VTG3、APOV1、RBP、RNF186。5条关键信号通路分别是脂肪消化吸收、胆固醇代谢、ECM-受体相互作用、雌激素信号通路、D-谷氨酰胺和D-谷氨酸代谢。综上,初步确定了6个康乐黄鸡开产性状相关候选基因,5条关键信号通路。

关键词: 康乐黄鸡, 肝脏, 候选基因, 关键通路, 转录组测序, 开产性状

Abstract:

The purpose of this study was to screen the candidate genes and key pathways related to traits at first laying through transcriptomic analysis of liver tissues on laying and non-laying Kangle yellow chickens,and to provide a theoretical basis for studying the molecular mechanisms of laying traits regulation in the liver of chickens.It also provides certain reference for the selection and breeding of Kangle yellow chickens.Three individuals of liver tissues at 154 days of age that had started laying (Group H) and had not started laying (Group L) were selected.Total RNA was extracted using the TRIzol method,and transcriptome sequencing was performed using the Illumina sequencing platform.Differentially expressed genes between the two groups were identified.The differentially expressed genes were subjected to functional enrichment and protein-protein interaction analysis.Nine candidate genes were randomly selected,and their expression levels were verified by qRT-PCR in the livers of the nine individuals.A total of 21 465 genes were detected to be expressed in the liver tissues,and a total of 227 differentially expressed genes were identified,among which 48 were up-regulated and 179 were down-regulated.The qRT-PCR validation results showed that the expression trends of nine genes in the two groups of individuals were basically consistent with the RNA-Seq results,and they showed a decreasing or increasing trend in the H,M(to be laid),and L three groups.Six candidate genes for the age at egg-laying trait were initially identified,namely VTG1,VTG2,VTG3,APOV1,RBP,and RNF186.The five crucial signaling pathways were fat digestion and absorption,cholesterol metabolism,ECM-receptor interaction,estrogen signaling pathway,D-glutamine and D-glutamate metabolism.Six genes and five key signaling pathways were preliminarily identified related to the traits at the first laying of Kangle yellow chicken.

Key words: Kangle yellow chicken, Liver, Candidate gene, Key pathway, Transcriptome sequencing, Egg-laying trait

中图分类号:

S831.2

马荆鄂, 曾配君, 万淑敏, 熊信威, 王樟凤, 刘婧, 许继国, 饶友生. 基于肝脏转录组测序筛选康乐黄鸡开产相关基因和关键通路[J]. 华北农学报, 2025, 40(3): 207-214. doi: 10.7668/hbnxb.20194898.

MA Jinge, ZENG Peijun, WAN Shumin, XIONG Xinwei, WANG Zhangfeng, LIU Jing, XU Jiguo, RAO Yousheng. Screening of Genes and Key Pathways Related to Egg-Laying in Kangle Yellow Chickens Based on Liver Transcriptome Sequencing[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 207-214. doi: 10.7668/hbnxb.20194898.

http://www.hbnxb.net/CN/Y2025/V40/I3/207

图/表 6

表1 引物序列信息

Tab.1 Primer sequence information

引物名称 Primer name	序列号 Accession numbers	引物序列(5'—3') Primer sequence(5'—3')		长度/bp Length
VTG1	NM_001004408.3	F:GAATTCCCGGGCAACTGCTA	R:TGCAAGTTTCTGCCTTGCTTC	157
VTG2	NM_001031276.2	F:ACAGCAAGGTCATTCGAGCA	R:CTCGGATGCCAACCTCTACC	171
VTG3	NM_001398307.1	F:TTGCTCAGTCGTGGGTCATC	R:CGAGTGTGGAGTCAGATGGG	216
APOV1	NM_205483.3	F:GAAAGCAGGACAGCAGGTCTC	R:GCCAGTCACGACGTTCTCTG	161
ITGB5	NM_204483.2	F:CCCGGCAGAGGTCTCAAC	R:GCTCAAGGGGACGTTCTTCA	225
RNF186	XM_004947482.5	F:TGCAGCATCTCTCGCTATGG	R:GAGCTTTGGGACCCGGTAAA	151
RBP	XM_046922434	F:GGCAGCTCAGTAAATCCTGTGA	R:CCGTCAGCCAGTTATGAGCA	195
LOC419888	XM_025143816.3	F:CTGCAACTGCTGTAGGACTCTT	R:GGCTCAGCAAGACAGTACCC	128
CETP	NM_001034814.3	F:ACGGTCACTATCCAAGCTGC	R:ACGGACTGGTCATTTCCACTT	119
β-actin	NM_205518.2	F:GCCGAGAGAGAAATTGTGCGTGAC	R:ACCACAGGACTCCATACCCAAGAAAG	210

表2 转录组测序基本特征

Tab.2 Basic features of transcriptome sequencing

样品 Sample	过滤后reads Clean reads	高质量(≥Q30)数据占比/% Ratio of high-quality(≥Q30)data	序列总数 Total reads	比对百分比/% Mapped reads percentage
H-LIV-1	21 558 777	94.33	43 177 554	92.64
H-LIV-2	20 277 431	92.96	40 554 862	92.00
H-LIV-3	24 731 384	94.14	49 462 768	92.17
L-LIV-1	23 403 738	94.97	46 807 476	93.57
L-LIV-2	21 418 718	94.89	4 287 436	92.27
L-LIV-3	21 699 178	94.31	43 398 356	92.20

图1 H组与L组差异表达基因GO注释分类横坐标为GO分类,纵坐标为基因数目,不同颜色代表所属不同的一级分类。1.单有机体过程;2.细胞过程;3.生物调节;4.代谢过程;5.多细胞有机体过程;6.刺激反应;7.发育过程;8.定位;9.信号;10.细胞组成、组织或生物发生;11.免疫系统过程;12.多生物体过程;13.繁殖;14.繁殖过程;15.行为;16.生长;17.运动;18.节律过程;19.解毒;20.生物黏附;21.参与化学突触传递的突触前过程;22.细胞;23.细胞组分;24.细胞器;25.膜;26.细胞器组分;27.膜组分;28.大分子复合物;29.膜封闭腔;30.胞外区;31.胞外区组分;32.其他有机体;33.其他有机体部分;34.突触部分;35.突触;36.超分子复合物;37.细胞连接;38.结合;39.催化活性;40.转运活性;41.分子转导活性;42.核酸结合转录因子活性;43.信号转导活性;44.分子功能调控;45.转录因子活性,蛋白结合;46.结构分子活性。

Fig.1 GO annotation classification of differentially expressed genes in group H and L The horizontal axis represents GO classification,while the vertical axis represents the number of genes,different colors represent different primary classifications.1.Single-organism process;2.Cellular process;3.Biological regulation;4.Metabolic process;5.Multicellular organismal process;6.Response to stimulus;7.Developmental process;8.Localization;9.Signaling;10.Cellular component organization or biogenesis;11.Immune system process;12.Multi-organism process;13.Reproduction;14.Reproductive process;15.Behavior;16.Growth;17.Locomotion;18.Rhythmic process;19.Detoxification;20.Biological adhesion;21.Presynaptic process involved in chemical synaptic transmission;22.Cell;23.Cell part;24.Organelle;25.Membrane;26.Organelle part;27.Membrane part;28.Macromolecular complex;29.Membrane-enclosed lumen;30.Extracellular region;31.Extracellular region part;32.Other organism;33.Other organism part;34.Synapse part;35.Synapse;36.Supramolecular complex;37.Cell junction;38.Binding;39.Catalytic activity;40.Transporter activity;41.Molecular transducer activity;42.Nucleic acid binding transcription factor activity;43.Signal transducer activity;44.Molecular function regulator;45.Transcription factor activity,protein binding;46.Structural molecule activity.

图2 H组与L组差异表达基因KEGG富集柱状图 1.脂肪消化吸收;2.胆固醇代谢;3.ECM-受体相互作用;4.雌激素信号通路;5.D-谷氨酰胺和D-谷氨酸代谢;6.丁酸代谢;7.鞘糖脂生物合成-乳糖和新乳糖系列;8.维生素的消化吸收;9.卟啉和叶绿素代谢;10.药物代谢-细胞色素P450;11.铁死亡;12.过氧化物酶体增殖物激活受体信号通路;13.细胞色素P450单加氧酶外源物质代谢;14.药物代谢-其他酶;15.逆行内源性大麻素信号;16.精氨酸和脯氨酸代谢;17.视黄醇代谢;18.核苷酸切除修复;19.溶酶体;20.谷胱甘肽代谢。

Fig.2 KEGG enrichment histogram of differentially expressed genes between group H and L 1.Fat digestion and absorption;2.Cholesterol metabolism;3.ECM-receptor interaction;4.Estrogen signaling pathway;5.D-Glutamine and D-glutamate metabolism;6.Butanoate metabolism;7.Glycosphingolipid biosynthesis-lacto and neolacto series;8.Vitamin digestion and absorption;9.Porphyrin and chlorophyll metabolism;10.Drug metabolism-cytochrome P450;11.Ferroptosis;12.PPAR signaling pathway;13.Metabolism of xenobiotics by cytochrome P450;14.Drug metabolism-other enzymes;15.Retrograde endocannabinoid signaling;16.Arginine and proline metabolism;17.Retinol metabolism;18.Nucleotide excision repair;19.Lysosome;20.Glutathione metabolism.

图3 H组与L组差异基因的蛋白互作网络图节点越大、颜色越深表示与其具有互作关系的基因越多。

Fig.3 Protein interaction network of differentially expressed genes between group H and L The gene with larger node and the darker color represented it interacted with more genes.

图4 候选基因qRT-PCR验证标注不同小写字母表示差异显著(P<0.05)。

Fig.4 qRT-PCR validation of candidate genes Different letters indicated a significant difference (P<0.05).

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