安格斯牛十字部高性状全基因组关联分析及<i>PSEN1</i>基因对成肌细胞增殖分化的影响

doi:10.7668/hbnxb.20194032

摘要/Abstract

摘要：

旨在筛选出与宁夏地区安格斯牛十字部高性状显著相关的单核苷酸多态性位点(SNPs)及对应候选基因,并探究干扰候选基因PSEN1对成肌细胞增殖分化的影响。以宁夏地区饲养的211头安格斯牛母牛为研究对象,通过全基因组关联分析,筛选与安格斯牛十字部高性状显著相关SNPs及对应候选基因;利用实时荧光定量PCR(qRT-PCR)检测候选基因在成肌细胞不同分化时期的表达规律;合成基因PSEN1的特异性干扰片段转染成肌细胞,利用qRT-PCR和EdU染色技术检测干扰PSEN1基因对成肌细胞的影响。结果表明:位于1,3,4,5,7,10,13,15,20,28号染色体的14个位点及相应13个候选基因与十字部高显著相关。在成肌细胞分化过程中,PSEN1基因的表达量呈先上升后下降的趋势,第4天达到最高。与对照组相比,干扰组阳性细胞率极显著低于对照组(P<0.01),肌肉发育标志基因PAX3、MEF2C、IGF2和MYOG的表达量显著降低(P<0.05),PCNA、MEF2B、MYH1、CDK1和MYF5等基因虽然差异不显著,但整体仍呈现下降趋势,表明干扰PSEN1基因后会抑制成肌细胞的增殖和分化。确定了影响安格斯牛十字部高性状的候选基因;PSEN1基因在成肌细胞增殖和分化过程中具有重要作用,可能是影响安格斯牛生长发育的一个潜在候选基因。

关键词: 安格斯牛, 十字部高, 全基因组关联分析, SNP, PSEN1, 成肌细胞

Abstract:

The purpose of this study was to screen single nucleotide polymorphisms(SNPs)and candidate genes significantly associated with high cross traits in Angus cattle in Ningxia,and to investigate the effect of interference with PSEN1 on the proliferation and differentiation of myogenic cells.A group of 211 Angus cows reared in Ningxia was used to screen SNPs and candidate genes significantly associated with high cross traits in Angus cows by genome-wide association analysis;the expression pattern of candidate genes at different differentiation periods of adult myoblasts was detected by Real-time Fluorescence Quantitative PCR(qRT-PCR);the specific interfering fragment of synthetic gene PSEN1 was transfected into adult myoblasts;qRT-PCR and EdU staining techniques were used to detect the effect of PSEN1 gene on myogenic cells.The results showed that 14 SNPs located on chromosomes 1,3,4,5,7,10,13,15,20 and 28 and the corresponding 13 candidate genes were significantly associated with high cross section.During myogenic cell differentiation,the expression of PSEN1 gene showed a trend of increasing and then decreasing,reaching the highest on the fourth day.Compared with the control group,the positive cell rate was extremely significantly lower in the interference group(P<0.01),the expression of muscle development marker genes PAX3,MEF2C,IGF2 and MYOG was significantly lower(P<0.05),and the genes PCNA,MEF2B,MYH1,CDK1 and MYF5 showed an overall decreasing trend although the differences were not significant,indicating that interference PSEN1 gene suppressed the proliferation and differentiation of myogenic cells.This study identified candidate genes affecting the cross height trait in Angus cattle,which provided a basis for the improvement of production performance and breed selection in Ningxia Angus cattle;PSEN1 gene had an important role in the proliferation and differentiation of adult myoblasts and might be a potential candidate gene affecting the growth and development of Angus cattle.

Key words: Angus cattle, Height cross, Genome-wide association analysis, SNP, PSEN1, Myogenic cells

李德生, 蒋秋斐, 封元, 王瑜, 冯小芳, 禹保军, 陈亚飞, 张迪, 王川川, 李蕾蕾, 顾亚玲, 张娟. 安格斯牛十字部高性状全基因组关联分析及PSEN1基因对成肌细胞增殖分化的影响[J]. 华北农学报, 2023, 38(5): 206-217. doi: 10.7668/hbnxb.20194032.

LI Desheng, JIANG Qiufei, FENG Yuan, WANG Yu, FENG Xiaofang, YU Baojun, CHEN Yafei, ZHANG Di, WANG Chuanchuan, LI Leilei, GU Yaling, ZHANG Juan. Genome-wide Association Analysis of Angus Cattle Cross Section Height Traits and the Effect of PSEN1 Gene on Proliferation and Differentiation of Myogenic Cells[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 206-217. doi: 10.7668/hbnxb.20194032.

http://www.hbnxb.net/CN/Y2023/V38/I5/206

图/表 14

参考文献 42

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基因 Gene	序列(5'-3') Primer sequence(5'-3')	退火温度/℃ Tm	产物片段/bp Product size
PSEN1	F:TGAGCACGGCAACGAG	54	146
	R:GCCACAACTACCACCATACA
PCNA	F:TGAAGAAAGTGCTGGAGGCG	60	157
	R:TTTTGGACATGCTGGTGAGG
PAX3	F:ACTGGAGCGGGCTTTTGA	55	198
	R:ATGGCTGTGGGAGGGAAT
MEF2C	F:TTGTGTGCTGTTCCACCTCCC	58	142
	R:GATTGCCATACCCGTTCCCTG
MEF2B	F:ATCGCCCTCATCATCTTC	57	146
	R:CTCCGCTTCAGTGTCTCG
IGF2	F:CGGTGCTGGTGCTTCTTGC	60	129
	R:CGTCGGTTTATGCGGCTGG
MYOG	F:TCTATCAGGAACCTCACT	58	194
	R:CTTACACACCTTACACGC
CDK1	F:AGAAAGTGAAGAGGAAGGGG	55	187
	R:TGAACTGACCAGGAGGGATA
MYH1	F:GCAGGGAAGGGGGGAAGGT	58	177
	R:GGAGAAGATGTGGGGCGGG
MYF5	F:GTTCGGGGACGAGTTTGA	56	158
	R:GCCTCTGGTTAGGGTTGG
GAPDH	F:TCACCAGGGCTGCTTTTA	58	126
	R:CTGTGCCGTTGAACTTGC

基因 Gene	序列(5'-3') Primer sequence(5'-3')	退火温度/℃ Tm	产物片段/bp Product size
PSEN1	F:TGAGCACGGCAACGAG	54	146
	R:GCCACAACTACCACCATACA
PCNA	F:TGAAGAAAGTGCTGGAGGCG	60	157
	R:TTTTGGACATGCTGGTGAGG
PAX3	F:ACTGGAGCGGGCTTTTGA	55	198
	R:ATGGCTGTGGGAGGGAAT
MEF2C	F:TTGTGTGCTGTTCCACCTCCC	58	142
	R:GATTGCCATACCCGTTCCCTG
MEF2B	F:ATCGCCCTCATCATCTTC	57	146
	R:CTCCGCTTCAGTGTCTCG
IGF2	F:CGGTGCTGGTGCTTCTTGC	60	129
	R:CGTCGGTTTATGCGGCTGG
MYOG	F:TCTATCAGGAACCTCACT	58	194
	R:CTTACACACCTTACACGC
CDK1	F:AGAAAGTGAAGAGGAAGGGG	55	187
	R:TGAACTGACCAGGAGGGATA
MYH1	F:GCAGGGAAGGGGGGAAGGT	58	177
	R:GGAGAAGATGTGGGGCGGG
MYF5	F:GTTCGGGGACGAGTTTGA	56	158
	R:GCCTCTGGTTAGGGTTGG
GAPDH	F:TCACCAGGGCTGCTTTTA	58	126
	R:CTGTGCCGTTGAACTTGC

阶段 Stage	样本量 Amount of data	均值/cm Mean	标准差 s	变异系数 CV	最大值/cm Max	最小值/cm Min
初生 Newborn	211	74.13	5.27	4.78	91	62
断奶 Weaning	211	98.36	5.88	5.98	106	80
6月龄 6 months old	211	107.18	6.34	5.05	130	91
12月龄 12 months old	211	119.26	8.19	6.87	136	106
18月龄 18 months old	211	127.99	8.70	6.80	146	116
24月龄 24 months old	211	134.18	8.64	6.21	148	117

阶段 Stage	样本量 Amount of data	均值/cm Mean	标准差 s	变异系数 CV	最大值/cm Max	最小值/cm Min
初生 Newborn	211	74.13	5.27	4.78	91	62
断奶 Weaning	211	98.36	5.88	5.98	106	80
6月龄 6 months old	211	107.18	6.34	5.05	130	91
12月龄 12 months old	211	119.26	8.19	6.87	136	106
18月龄 18 months old	211	127.99	8.70	6.80	146	116
24月龄 24 months old	211	134.18	8.64	6.21	148	117

阶段 Stage	数量 Amount	3'端非翻译区 3'-UTR	5'端非翻译区 5'-UTR	下游区域 Down stream	外显子区域 Exonic region	基因间区 Intergenic	内含子区域 Intronic region	非编码RNA内含子区域 Non-coding RNA intron	基因上游区域 Up stream
初生 Newborn	52	0	0	0	0	30	22	0	0
断奶 Weaning	2	0	0	0	0	1	1	0	0
6月龄 6 months old	108	0	1	1	0	70	36	0	0
12月龄 12 months old	580	2	0	3	2	369	196	1	7
18月龄 18 months old	732	3	2	10	6	401	306	0	4
24月龄 24 months old	7	0	0	0	0	3	4	0	0
总计 Total	1 481	5	3	14	8	874	565	1	11

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安格斯牛十字部高性状全基因组关联分析及PSEN1基因对成肌细胞增殖分化的影响

Genome-wide Association Analysis of Angus Cattle Cross Section Height Traits and the Effect of PSEN1 Gene on Proliferation and Differentiation of Myogenic Cells

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模型 Model	阶段 Stage	数量 Amount	显著相关的位点数量 Number of significantly correlated loci	潜在相关的位点数量 Number of potentially relevant loci
MLM模型	初生	52	17	35
Mixed Linear	断奶	2	0	2
Model	6月龄	108	18	90
	12月龄 12	580	397	183
	18月龄 18	732	428	304
	24月龄 24	7	0	7
	总计	1 481	860	621

候选基因名称 Gene name	登录号 Registration number	描述 Description	条目类型 Item type
PSEN1	GO:0045821	糖酵解正向调节	生物学过程
	GO:0060828	规范Wnt信号通路的调控	生物学过程
	GO:0043066	细胞凋亡过程的负调节	生物学过程
	GO:0048705	骨骼系统形态发生	生物学过程
	GO:0045296	钙粘蛋白结合	分子功能
	GO:0008013	β-连环蛋白结合	分子功能
	GO:0009986	细胞表面	细胞组分
	GO:0005887	质膜的组成部分	细胞组分
	GO:0030424	轴突	细胞组分
EYA2	GO:0014706	横纹肌组织发育	生物学过程
	GO:0030154	细胞分化	生物学过程
	GO:0006470	蛋白质去磷酸化	生物学过程
	GO:0000287	镁离子结合	分子功能
	GO:0005634	核	细胞组分
	GO:0005829	细胞质溶胶	细胞组分
IL6ST	GO:0045669	成骨细胞分化的正向调节	生物学过程
	GO:0008593	Notch信号通路的调节	生物学过程
	GO:0042102	T细胞增殖的正向调节	生物学过程
	GO:0005138	白细胞介素-6受体结合	分子功能
	GO:0043025	神经元细胞体	细胞组分
	GO:0043235	受体复合物	细胞组分
MEGF10	GO:0022409	细胞间黏附的正向调节	生物学过程
	GO:2000288	肌母细胞增殖的积极调节	生物学过程
	GO:0051451	肌母细胞迁移	生物学过程
	GO:0005112	Notch 结合	分子功能
	GO:0005044	清道夫受体活性	分子功能
	GO:0016021	膜的组成部分	细胞组分
ZHX3	GO:0045669	成骨细胞分化的正向调节	生物学过程
	GO:0045892	转录DNA模板化的负调节	生物学过程
	GO:0003677	脱氧核糖核酸结合	分子功能
	GO:0005634	核	细胞组分
ZMIZ1	GO:0007179	转化生长因子β受体信号通路	生物学过程
	GO:0048146	成纤维细胞增殖的正向调节	生物学过程
	GO:0046332	SMAD绑定	分子功能
	GO:0008270	锌离子结合	分子功能
	GO:0005654	核质	细胞组分
	GO:0005829	细胞质溶胶	细胞组分
IGF1	GO:0045821	糖酵解正向调节	生物学过程
	GO:0045445	肌母细胞分化	生物学过程
	GO:0060416	对生长激素的反应	生物学过程
	GO:0043066	细胞凋亡过程的负调节	生物学过程
	GO:0045669	成骨细胞分化的正向调节	生物学过程
	GO:0048146	成纤维细胞增殖的正向调节	生物学过程
	GO:0005158	胰岛素受体结合	分子功能
	GO:0005829	胞浆	细胞组分
SHH	GO:0030336	细胞迁移的负调控	生物学过程
	GO:0030177	Wnt信号通路的正向调节	生物学过程
	GO:0014706	横纹肌组织发育	生物学过程
	GO:0048745	平滑肌组织发育	生物学过程
	GO:0005509	钙离子结合	分子功能
	GO:0005539	糖胺聚糖结合	分子功能
	GO:0009986	细胞表面	细胞组分
PDGFD	GO:0043406	MAP激酶活性的正向调节	生物学过程
	GO:0030335	细胞迁移的正向调控	生物学过程
	GO:0031954	蛋白质自磷酸化的正向调节	生物学过程
	GO:0070851	生长因子受体结合	分子功能
	GO:0016020	膜	细胞组分

染色体 Chromosome	物理位置/bp Position	候选基因 Gene	染色体 Chromosome	物理位置/bp Position	候选基因 Gene
10	84 699 323	PSEN1	5	66 589 147	IGF1
13	75 652 089	EYA2	4	117 584 718	SHH
20	23 205 273	IL6ST	15	4 231 029	PDGFD
7	26 471 248	MEGF10	3	14 343 331	MEF2D
13	69 914 824	ZHX3	7	1 579 250	MAML1
13	69 897 502		1	2 776 850	PAXBP1
28	34 817 449	ZMIZ1	28	17 991 101	ARID5B

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