基于垂体全长转录组测序分析鸡睾丸性状相关基因

doi:10.3864/j.issn.0578-1752.2024.20.017

Abstract

Abstract:

【Background】Testis is an important reproductive organ of male animals, which has the function of producing sperm and secreting androgen. The quality of testis is directly related to reproductive performance. Testicular traits are regulated by multiple genes in the hypothalamic-pituitary-gonadal axis, but the molecular mechanism is still unclear. The variation coefficient of testicular traits was large in Ningdu yellow chicken, with poor reproductive performance consistency. 【Objective】 In order to explore candidate genes and key signaling pathways, full-length transcriptome sequencing was performed on pituitary tissues of six individuals with different testicular traits. The selection and breeding of testis traits was expected to develop local chicken breeding with these founding genetic basis.【Method】A total of seventy 22-week-old Ningdu yellow roosters were studied. Six individuals were randomly selected, including three individuals with large testicular and others with small testicular. Full-length transcript expression profile was constructed on pituitary tissue for each individual. Then the genes or transcripts were selected with high expression or different expression between two groups of individuals. Gene function enrichment analysis was also performed for these differentially expressed genes or transcripts. Six genes were randomly selected to verify the gene expression level. Referred to known marker genes in pituitary cells, screening the differentially expressed genes or transcripts were carried out corresponding to the marker genes in the eight kinds of cells. The correlation was analyzed between testicular traits and these genes or transcripts and also those with high expressional level.【Result】In six pituitary tissues, 21 834 genes and 29 355 full-length transcripts were detected. Among them, 332 genes and 229 transcripts were differentially expressed between the two groups. KEGG pathway analysis showed that differentially expressed genes or transcripts were mainly concentrated in apelin signaling pathway, and glycerophospholipid metabolism. Five marker genes of the gonadotropin cell were identified related to part of testicular traits, including NFASC (neurofascin) and its transcript XM_015298904.2, TESC(tescalcin) and its transcript XM_025155510.1, ONT.20974.4 (one transcript of follicle stimulating hormone beta), RLN3 (relaxin 3), ONT.1176.4 (one transcript of NADH: ubiquinone oxidoreductase subunit B2). Four genes were also identified related to part of testicular traits, including EGR1 (early growth response 1) as one of the marker genes in the lactotrophs, TMSB4X (thymosin beta 4, X-linked) and C1H12ORF57(chromosome 1 open reading frame) as the marker genes of the folliculo-stellate cells, CHGB (chromogranin B) as one of the marker genes in the thyrotrophs. Two highly expressed genes were identified related to part of testicular traits, including TMSB4X and HSP90AA1 (heat shock protein 90 alpha family class A member 1). FSHB and EGR1 were found as node genes in the gene interaction network.【Conclusion】Preliminary functional analysis showed that EGR1, TMSB4X, FSHB and RLN3 were the key genes in testicular trait regulation. MAPK, neuroactive ligand-receptor interaction and GnRH signaling pathway played the key role in the regulation of testicular traits.

Key words: transcriptome sequencing, Ningdu yellow chicken, testicular quality, candidate gene

MA JingE, XIONG XinWei, ZHOU Min, WU SiQi, HAN Tian, RAO YouSheng, WANG ZhangFeng, XU JiGuo. Full-Length Transcriptomic Analysis of Chicken Pituitary Reveals Candidate Genes for Testicular Trait[J].Scientia Agricultura Sinica, 2024, 57(20): 4130-4144.

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基因名 Gene name	上游引物 Forward primer	下游引物 Reverse primer
WNT4	CATCGAGGAGTGCCAGTACC	TCGCATCCACACTTGTCCAG
GH	TGCCGAGACATATAAAGAGTTC	GAGCTGGGATGGTTTCTGAGTA
POMC	CCATAGCTCCTCCGCAGTTT	CCACGCTCCCACTTACCAAA
TPH1	TCTTGCATCACTTGGGGCAT	GCGAGTGCTTGAGCTCACTA
TSHB	ACTGCCTGGCCATCAACAC	ACACGTTTTGAGACAGAGCACTTTT
COCH	TTCATCTTTCTCAGTGACTCCAGG	AACATGAGGCCCTTCCGTTC
β-actin	TCATTGTGCTAGGTGCCA	CCTCTTCCAGCCATCTTT

性状 Traits	最小值 Minus	最大值 Max	平均值±标准差 Mean ± SD	变异系数 Variation coefficient
左侧睾丸质量 Left testis weight (g)	3.4	35.3	16.67±6.45	0.39
右侧睾丸质量 Right testis weight (g)	2.6	32.9	15±6.31	0.42
睾丸总质量 Total testis weight (g)	6.0	61.2	31.67±12.45	0.39
左睾丸系数 Left testis percentage	0.002	0.09	0.01±0.01	0.37
右睾丸系数 Right testis percentage	0.0017	0.06	0.01±0.01	0.40
睾丸系数 Total testis percentage	0.0037	0.15	0.02±0.02	0.37
屠宰活重 Body weight (g)	1776	2196	1778.76±276.47	0.11

组别 Group	屠宰活重 Body weight (g)	左侧睾丸质量 Left testis weight (g)	右侧睾丸质量 Right testis weight (g)	睾丸总质量 Total testis weight (g)	左睾丸系数 Left testis percentage	右睾丸系数 Right testis percentage	睾丸系数 Total testis percentage	屠宰活重平均值±标准差 Mean ± SD of the body weight	睾丸总质量平均值±标准差 Mean ± SD of the total testis weight
大睾丸组 H-PIT	1548.5	27.6	32.9	60.5	0.0180	0.0210	0.0390	1827±199.0	58.2±3.8*
	1931.4	23.9	28.9	52.8	0.0120	0.0150	0.0270
	2001.0	35.3	25.9	61.2	0.0180	0.0130	0.0310
小睾丸组 L-PIT	1586.1	5.1	4.1	9.2	0.0032	0.0026	0.0058	1432±113.4	7.8±1.4
	1316.9	4.5	3.8	8.3	0.0034	0.0029	0.0063
	1392.0	3.4	2.6	6.0	0.0024	0.0019	0.0043

转录本编号 Transcript ID	对应基因名 Gene name	H-PIT组平均CPM值 The mean CPM of the H-PIT	L-PIT组平均CPM值 The mean CPM of the L-PIT	6个样品平均CPM值 The mean CPM of the six individuals
ONT.45201.8	ONT.45201	59338.9	54252.0	56795.5
XM_421330.6	CHGA	49840.5	53654.7	51747.6
XM_015285103.2	POMC	39287.1	38699.3	38993.2
ONT.14667.8	CGA	36811.5	24317.2	30564.4
ONT.9069.1	PRL	26192.1	22811.4	24501.7
NM_204359.2	GH	14833. 4	27171.4	21002.4
NM_205500.2	CST3	13824.6	12563.8	13194.2
XM_004946664.3	UBB	7980.8	6959.8	7470.3
ONT.14667.1	ONT.14667	5623.5	8094.4	6858.9
ONT.39062.2	PPIA	5717.97	4953.3	5335.7

转录本编号 Transcript ID	基因名 Gene name	基因描述 Gene description
XM_420332.6	THOC2	THO复合体2 THO complex 2
XM_015299935.2	CIRBP	低温诱导RNA结合蛋白 cold inducible RNA binding protein
XM_025147888.1	GREB1L	GREB1样视黄酸受体共激活剂 GREB1 like retinoic acid receptor coactivator
XM_025154119.1	SCAPER	S期细胞周期素A相关蛋白 S-phase cyclin A associated protein in the ER
NM_001305114.2	ARL6	类ADP核糖化因子GTPase 6 ADP ribosylation factor like GTPase 6
NM_001012919.2	DNPEP	天门冬氨酰氨肽酶 Aspartyl aminopeptidase
ONT.28408.11	OTOS	耳螺菌素 Otospiralin
XM_421638.6	ABCG2	ATP结合盒G亚族成员2 ATP binding cassette subfamily G member 2
ONT.45513.10	TRIR	19号染色体开放阅读框43 Chromosome 19 open reading frame 43
XM_015298649.2	FDPS	法尼基二磷酸合成酶 Farnesyl diphosphate synthase

Full-Length Transcriptomic Analysis of Chicken Pituitary Reveals Candidate Genes for Testicular Trait

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转录本编号 Transcript ID	基因名 Gene name	基因描述 Gene description
XM_025149862.1	THOC2	THO复合体2 THO complex 2
XM_004940000.3	NUDCD1	NudC结构域1 NudC domain containing 1
NM_001278016.1	LYRM2	LYR 模块2 LYR motif containing 2
XM_015276855.2	GIGYF2	GRB10互作GYF蛋白2 GRB10 interacting GYF protein 2
XM_004935722.3	AHI1	Abelson助手集成点1 Abelson helper integration site 1
XM_001232161.4	ADAR	RNA特异性腺苷脱氨酶 Adenosine deaminase, RNA specific
XM_015290097.2	HSP40	热应激蛋白40 Heat shock protein 40
XM_015296842.2	AGRN	集聚蛋白 Agrin
ONT.45513.1	TRIR	19号染色体开放阅读框43 Chromosome 19 open reading frame 43
ONT.37858.1	WFDC2L	WAP四二硫核结构域样蛋白2 WAP four-disulfide core domain protein 2-like

基因名 Gene name	基因描述 Gene description	细胞类型 Cell type	染色体号 Chromosome No.	平均CPM值 The mean CPM	表达量上/下调 Up/down regulated
TFPI2	组织因子途径抑制物2 Tissue factor pathway inhibitor 2	Endo	2	39.01	up
TMSB4X	胸腺素β4X Thymosin beta 4, X-linked	FS	1	3765.66	down
C1H12ORF57	1号染色体开放阅读框人类C12orf57 Chromosome 1 open reading frame, human C12orf57	FS	1	32.85	up
MRPS12	线粒体核糖体蛋白S12 Mitochondrial ribosomal protein S12	FS	32	29.37	up
NFASC	神经束蛋白 Neurofascin	Gona	26	398.44	down
RLN3	松弛素3 Relaxin 3	Gona	Z	3.9	down
TESC	钙结合蛋白 Tescalcin	Gona	15	350.5	down
NDUFB2	NADH: 泛醌氧化还原酶B2亚基 NADH: ubiquinone oxidoreductase subunit B2	Gona	1	157.41	up
BTG2	抗增殖因子2 BTG anti-proliferation factor 2	Lac	26	234.04	up
DUSP1	双特异性磷酸酶1 Dual specificity phosphatase 1	Lac	13	51.76	up
EGR1	早期生长反应1 Early growth response 1	Lac	13	120.79	up
GPX1	谷胱甘肽过氧化物酶1 Glutathione peroxidase 1	RBC	12	117.38	up
CHGB	嗜铬粒蛋白B Chromogranin B	Thy	3	2338.41	up
DIO2	去碘酶，碘甲状腺原氨酸II型 Deiodinase, iodothyronine type II	Thy	5	134.46	up
SPCS3	信号肽酶复合体亚基3 Signal peptidase complex subunit 3	WBC	4	80.44	up

转录本编号 Transcript ID	对应基因名 Gene name	基因描述 Gene description	细胞类型 Cell type	染色体号 Chromosome No.	平均CPM值 The mean CPM	表达量上/下调 Up/down regulated
ONT.1176.4	NDUFB2	NADH: 泛醌氧化还原酶B2亚基 NADH: ubiquinone oxidoreductase subunit B2	Gona	1	95.7	上调Up
XM_015298904.2	NFASC	神经束蛋白 Neurofascin	Gona	26	1.4	下调Down
XM_025155510.1	TESC	钙结合蛋白 Tescalcin	Gona	15	149.8	下调Down
ONT.20974.4	FSHB	促卵泡激素亚基 Follicle stimulating hormone beta subunit	Gona	5	111.4	下调Down
ONT.34028.6	ACTB	β-肌动蛋白 Actin, beta	Lac	14	7.2	上调Up
XM_015294201.2	HAGH	羟酰谷胱甘肽水解酶 Hydroxyacylglutathione hydrolase	RBC	14	1.5	上调Up

基因名/转录本编号 Gene name/ transcript ID	屠宰重 Body weight	左睾丸重 Left testis weight	右睾丸重 Right testis weight	睾丸总重 Total testis weight	左睾丸率 Left testis percentage	右睾丸率 Right testis percentage	总睾丸率 Total testis percentage
NFASC	0.6	0.94**	0.94**	0.94**	0.94**	0.89*	0.94**
RLN3	0.7	0.8	0.8	0.8	0.8	0.83*	0.8
TESC	0.5	0.83*	1.0**	0.83*	0.89*	0.94**	0.89*
TMSB4X	0.4	0.89*	0.89*	0.89*	1.0**	0.94**	1.0**
CHGB	-0.6	-0.8	-0.94**	-0.8	-0.8	-.083*	-0.8
C1H12ORF57	-0.7	-0.829*	-0.7	-0.83*	-0.7	-0.6	-0.7
EGR1	-0.6	-0.8	-0.94**	-0.8	-0.8	-0.83*	-0.8
GPX1	-0.7	-0.83*	-0.7	-0.83*	-0.7	-0.6	-0.7
TFPI2	-0.94**	-0.89*	-0.7	-0.89*	-0.7	-0.6	-0.7
ONT.1176.4	-0.4	-0.7	-0.7	-0.7	-0.83*	-0.8	-0.83*
XM_015298904.2	0.4	0.6	0.81*	0.6	0.8	0.81*	0.8
XM_025155510.1	0.8	1.0**	0.83*	1.0**	0.89*	0.8	0.89*
ONT.20974.4	0.83*	0.83*	0.83*	0.83*	0.7	0.8	0.7
HSP90AA1	0.79	0.84*	0.75	0.81	0.82*	0.7	0.77

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