基于转录组与一代测序技术挖掘ADSL基因调控坝上长尾鸡肌苷酸含量

doi:10.3969/j.issn.1004-1524.20240222

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (12): 2676-2686.DOI: 10.3969/j.issn.1004-1524.20240222

基于转录组与一代测序技术挖掘ADSL基因调控坝上长尾鸡肌苷酸含量

何玲钰¹(), 乔贤¹^,², 王新越¹, 李祥龙¹^,²^,^*()

1.河北科技师范学院动物科技学院,河北秦皇岛 066000
2.河北省特色动物种质资源挖掘与创新重点实验室,河北秦皇岛 066000

收稿日期:2024-03-08 出版日期:2024-12-25 发布日期:2024-12-27
作者简介:何玲钰(1999—),女,河北石家庄人,硕士研究生,研究方向为动物遗传育种。E-mail:h19943450526@163.com
通讯作者: *李祥龙,E-mail:15203358192@163.com
基金资助:
河北省鸡现代种业科技创新团队(21326303D)

Exploring ADSL gene regulation of inosine monophosphate content in Bashang long tail chicken based on transcriptome and next-generation sequencing technology

HE Lingyu¹(), QIAO Xian¹^,², WANG Xinyue¹, LI Xianglong¹^,²^,^*()

1. College of Animal Science and Technology, Hebei Normal University of Science and Technology, Qinhuangdao 066000, Hebei, China
2. Hebei Provincial Key Laboratory of Characteristic Animal Germplasm Resources Mining and Innovation, Qinhuangdao 066000, Hebei, China

Received:2024-03-08 Online:2024-12-25 Published:2024-12-27

摘要/Abstract

摘要：

为寻找与改良坝上长尾鸡肌肉肌苷酸(inosine monophosphate,IMP)含量的基因,试验选取河北省优质蛋肉兼用型品种坝上长尾鸡为对象,对60只进行解剖测得肌苷酸含量,以坝上长尾鸡肌苷酸含量高低为指标分组,利用转录组测序进行差异表达基因分析和KEGG通路富集,并利用PCR扩增和测序技术,进行多态位点的筛查,使用SPSS中的单因素方差分析与肌苷酸含量进行关联分析。结果表明,转录组测序分析中差异显著表达基因中有251个上调基因、241个下调基因,另有18 300个差异不显著,对差异基因进行KEGG通路富集分析,发现核苷酸代谢途径对肌苷酸的含量有着重要影响,ADSL基因参与该通路最终影响肌苷酸的合成。成功扩增ADSL基因第2、7、9、11外显子,筛查到g.3713G>A,g.3797C>T,g.8282C>G,g.10191C>T,g.12827C>A,其中外显子2(g.3797C>T)CT型IMP含量显著(P<0.05)高于CC型与TT型,外显子7(g.8282C>G)CG型IMP含量显著(P<0.05)高于GG型及CC型,说明坝上长尾鸡ADSL基因第2外显子g.3797C>T位点CT型及第7外显子g.8282C>G位点CG型为优势基因型,可作为IMP含量的分子标记用于育种实践。

关键词: 坝上长尾鸡, 转录组测序, ADSL基因, 多态位点, 肌苷酸

Abstract:

In order to search and improve the genes involved in the muscle inosine monophosphate (IMP) content of Bashang long tail chickens, a high-quality meat and egg dual-use breed of Bashang long tail chickens in Hebei Province was selected as the experimental subjects. The inosine content of 60 chickens was measured. The high and low inosine monophosphate content of Bashang long tail chickens was used as the indicator for grouping, and transcriptome sequencing was performed for differential expression gene analysis and KEGG pathway enrichment. PCR amplification and sequencing technology were used to screen for polymorphic sites. Use one-way ANOVA in SPSS to conduct correlation analysis with inosine monophosphate content. The results showed that:among the significantly differentially expressed genes in transcriptome sequencing analysis, there were 251 up-regulated genes and 241 down-regulated genes, while 18 300 genes were not significantly different. KEGG pathway enrichment analysis was conducted on the differentially expressed genes, and it was found that the nucleotide metabolism pathway has an important impact on the content of inosine monophosphate. The ADSL gene is involved in this pathway and ultimately affects the synthesis of inosine monophosphate. the 2nd, 7th, 9th, and 11th exons of the ADSL gene were successfully amplified, and g. 3713G>A, g. 3797C>T, g. 8282C>G, g. 10191C>T, and g. 12827C>A were screened. Among them, the IMP content in exon 2 (g. 3797C>T) CT individuals was significantly (P<0.05) higher than that in CC and TT individuals, while in exon 7 (g. 8282C>G), the IMP content in CG individuals was significantly (P<0.05) higher than that in GG individuals and CC individuals. The dominant genotypes of the ADSL gene in Bashang long tail chickens are located at exon 2, g. 3797C>T site CT type, and exon 7, g. 828282C>G site CG type, which can be used as molecular markers for IMP content in breeding practice.

Key words: Bashang long tail chicken, transcriptome sequencing, ADSL, polymorphic locus, inosine monophosphate

中图分类号:

S831

何玲钰, 乔贤, 王新越, 李祥龙. 基于转录组与一代测序技术挖掘ADSL基因调控坝上长尾鸡肌苷酸含量[J]. 浙江农业学报, 2024, 36(12): 2676-2686.

HE Lingyu, QIAO Xian, WANG Xinyue, LI Xianglong. Exploring ADSL gene regulation of inosine monophosphate content in Bashang long tail chicken based on transcriptome and next-generation sequencing technology[J]. Acta Agriculturae Zhejiangensis, 2024, 36(12): 2676-2686.

图/表 14

参考文献 31

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样品编号(A组) Sample name (Group A)	肌苷酸含量 Inosine monophosphate content/(mg·kg^-1)	样品编号(B组) Sample name (Group B)	肌苷酸含量 Inosine monophosphate content/(mg·kg^-1)
1	1 183.73	6	11.28
2	1 601.84	7	17.71
3	1 173.14	8	12.43
4	1 246.67	9	13.21
5	1 153.34	10	14.01

样品编号(A组) Sample name (Group A)	肌苷酸含量 Inosine monophosphate content/(mg·kg^-1)	样品编号(B组) Sample name (Group B)	肌苷酸含量 Inosine monophosphate content/(mg·kg^-1)
1	1 183.73	6	11.28
2	1 601.84	7	17.71
3	1 173.14	8	12.43
4	1 246.67	9	13.21
5	1 153.34	10	14.01

基因 Gene	SNP位点 SNP site	引物序列 Primer sequences(5'→3')	产物大小 Product size/bp	退火温度 Annealing temperature/℃
ADSL(AY665559.1)	37133797	F:CTTTTGACTGCCCCATTGCC R:AAAGGGAGAAGCCACAGGAG	278	57.2
	8282	F:CTTTCATTCCCACTTGCGGC R:GCTTGGACCAATTCAGCACA	333	56.0
	10191	F:TCCCCACCGCATTTAGATGT R:GTCTGCTTTTCATCAAGACGGT	192	56.4
	12827	F:TCCACAGTAACGTTCCAGACA R:TTCACAGAAGGAAAGCGCCT	341	56.9

基因 Gene	SNP位点 SNP site	引物序列 Primer sequences(5'→3')	产物大小 Product size/bp	退火温度 Annealing temperature/℃
ADSL(AY665559.1)	37133797	F:CTTTTGACTGCCCCATTGCC R:AAAGGGAGAAGCCACAGGAG	278	57.2
	8282	F:CTTTCATTCCCACTTGCGGC R:GCTTGGACCAATTCAGCACA	333	56.0
	10191	F:TCCCCACCGCATTTAGATGT R:GTCTGCTTTTCATCAAGACGGT	192	56.4
	12827	F:TCCACAGTAACGTTCCAGACA R:TTCACAGAAGGAAAGCGCCT	341	56.9

样品名称 Sample name	浓度 Concentration/ (ng·μL^-1)	D₂₆₀/D₂₈₀	RNA完整值 RNA integrity number(RIN)
A1	139	2.006	6.8
A2	156	2.012	7.5
A3	128	2.000	8.2
A4	132	1.982	7.9
A5	152	2.003	8.3
B1	161	2.036	7.7
B2	137	1.908	8.5
B3	166	2.016	8.3
B4	159	2.027	9.1
B5	118	1.968	8.6

基于转录组与一代测序技术挖掘ADSL基因调控坝上长尾鸡肌苷酸含量

Exploring ADSL gene regulation of inosine monophosphate content in Bashang long tail chicken based on transcriptome and next-generation sequencing technology

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样本 Sample	原始reads Raw reads	过滤后reads Clean reads	过滤后碱基 Clean bases/Gb	错误发现率 False discovery rate/%	Q20%	Q30%	GC含量 GC content/%
A1	50 728 000	48 889 916	7.33	0.03	97.39	93.09	51.85
A2	42 785 508	40 886 724	6.13	0.03	97.28	92.85	51.58
A3	47 588 060	45 744 878	6.86	0.03	97.19	92.63	52.19
A4	52 503 284	50 632 380	7.59	0.03	96.72	91.73	50.37
A5	43 447 046	41 692 442	6.25	0.03	97.44	93.39	52.31
B1	46 702 784	44 980 912	6.75	0.03	97.29	92.8	52.23
B2	41 813 830	40 168 072	6.03	0.03	97.28	92.84	52.61
B3	43 513 932	42 487 038	6.37	0.03	96.88	91.9	52.94
B4	45 702 690	44 466 824	6.67	0.03	97.03	92.29	50.88
B5	46 914 640	45 820 392	6.87	0.03	97.37	93.1	52.39

SNP位点 SNP site	基因型频率(个体数) Genotype frequency							基因频率 Allele frequency				PIC	χ²	P值 P value
	AA	AG	GG	CG	CC	CT	TT	A	G	C	T
g.3713G>A	0.61(32)	0.30(16)	0.09(5)					0.75	0.25			0.30	15.07	0.00
g.3797C>T					0.54(28)	0.15(8)	0.31(16)			0.61	0.39	0.36	23.82	—
g.8282C>G			0.46(25)	0.39(21)	0.15(8)				0.66	0.34		0.35	1.48	0.48
g.10191C>T					0.92(49)	0.08(4)				0.96	0.04	0.07	1.00	—
g.12827C>A	0.06(3)				0.94(50)			0.06		0.94		0.11	5.33	0.07

SNP位点 SNP site	基因型 Genotype	样本 Sample size	肌苷酸含量 IMP content/(g·kg^-1)
g.3713G>A	AA	32	2.2±3.5
	AG	16	4.2±4.8
	GG	5	1.1±1.2
g.3797C>T	CC	28	2.1±3.2 a
	CT	8	6.3±6.0 b
	TT	16	2.1±3.1 a
g.8282C>G	CC	8	2.1±2.2 a
	CG	21	4.2±5.1 b
	GG	25	1.5±2.4 a
g.10191C>T	CC	49	2.8±4.0
	CT	4	1.4±1.1
g.12827C>A	AA	3	4.3±3.6
	CC	50	2.6±3.9

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