海南黑山羊ATG16L2基因启动子区多态性研究

doi:10.11843/j.issn.0366-6964.2024.11.017

Abstract

Abstract:

The study aimed to investigate the structural characteristics and genetic distribution of the ATG16L2 gene promoter region in Hainan Black goat, providing a theoretical basis for further exploring the expression regulation mechanism and function of this gene. This study focused on 200 Hainan Black goats, constructing a DNA pooling strategy. The polymorphism of the ATG16L2 gene promoter region in Hainan Black goats was initially screened using Sanger sequencing. PCR-RFLP technology was applied for genotyping the 200 Hainan Black goats. Linkage disequilibrium analysis was performed on the identified SNP loci to construct haplotypes. Bioinformatics methods were utilized to analyze the impact of SNP loci on the expression of the ATG16L2 gene in Hainan Black goats. The promoter region of the ATG16L2 gene in Hainan Black goats contained 3 linked single nucleotide polymorphisms (SNPs): SNP1 (g.30667970T > C), SNP2 (g.30668540T > C), and SNP3 (g.30668664C > T). Both SNP1 and SNP2 exhibited moderate levels of polymorphism, whereas SNP3 showed low levels. Notably, all three SNPs conformed to the principles of Hardy-Weinberg equilibrium(P > 0.05). The haplotype analysis revealed that the frequencies of haplotype H1, H2, H3, and H4 were 0.321, 0.304, 0.271, and 0.097, respectively, with H1 (CGC) being the predominant haplotype. The bioinformatics analysis results showed that there were 3 promoters, 4 CpG island regions, 2 repetitive elements LINE2 (—1 989-—1 826 bp, —562-—426 bp), hAT-Charlie (—1 804-—1 511 bp), and 5 CCAAT-Box, 13 CAAT-Box, 10 CGCG-Box, 11 GATA-Box and 2 TATA-Box of the ATG16L2 gene in Hainan Black goat. The integrated prediction from various online tools suggests that the above SNPs may affect the expression regulation of the ATG16L2 gene in Hainan Black goats by influencing the changes in transcription factors in the gene's promoter region. In this study, 3 SNP loci were identified in the promoter sequence of the ATG16L2 gene in Hainan Black goats. Among these, SNP1 and SNP2 exhibited moderate polymorphism, while SNP3 showed low polymorphism. It is predicted that these SNPs may influence transcription factor binding, thereby regulating gene expression. This provides a theoretical basis for further exploration of the function and regulatory mechanisms of the ATG16L2 gene.

Key words: Hainan Black goat, ATG16L2 gene, promoter, polymorphism, bioinformatics

CLC Number:

S827.2

Huan WANG, Taoyu CHEN, Hui WU, Yong MENG, Shiyuan LI, Hejie QIAN, Shihua NIU, Churiga MAN, Qiaoling CHEN, Hongyan GAO, Li DU, Fengyang WANG, Si CHEN. Polymorphism Analysis of the ATG16L2 Gene Promoter Region in Hainan Black Goat[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 4980-4991.

Figures/Tables 13

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References 39

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名称Name	上游引物(5′→3′) Forward primer	下游引物(5′→3′) Reverse primer	产物长度/bp Length	退火温度/℃ Annealing temperature
P1	TGGGCAGGAGCCAGACTAAGC	AAGGGGATGGTACGCATGGAG	668	63.2
P2	CGGCTAGCGGAAATCCTGACCCCAATATGAC	CAGCTGATTTCATCGAGCAAACCCC	847	62.0
P3	GGCTAGCGCTCCACTGAATCACTCCTT	TCTGGACCAGGCGGTGCACTAAACT	658	62.0
P4	GGCTAGCGCTGCCTGGTATCCGTTTC	CCTCGAGATAGGGCCTTTTGCGTTCGATCC	729	62.0
P5	CAGCAGCATACACGCTCCTGCG	GCTGAGCCGGCCAGCAGCT	294	66.6
P6	ATCCTGACCCCAATATGACTC	ATGGTACGCATGGAGGATC	198	55.3
P7	ATTGGTCATCACTTCATGGCTTTCT	CATATTGTCAAGGCTTTGCCTTCAT	317	60.0

软件分析Software analysis	网站Website
启动子序列的特征分析Characteristic analysis of promoter sequences	https://services.healthtech.dtu.dk/services/Promoter-2.0/
重复元件分析Repeat element analysis	https://www.repeatmasker.org/cgi-bin/WEBRepeatMasker
顺式作用元件分析Analysis of cis-acting elements	https://www.dna.affrc.go.jp/PLACE/?action=newplace
CpG岛位点分析CpG island site analysis	http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi
转录因子结合预测Transcription factor binding prediction	http://jaspar.generec.net/
转录因子结合预测Transcription factor binding prediction	http://bioinfo.life.hust.edu.cn/AnimalTFDB4/#/TFBS_Predict

物种Species	相似性/% Similarity	物种Species	相似性/% Similarity
羚羊Budorcas taxicolor	99.03	绵羊Ovis aries	98.64
普通牛Bos taurus	96.81	瘤牛Bos indicus	91.61
猪Sus scrofa	86.87	马Equus caballus	86.51
雪貂Mustela putorius furo	82.71	老虎Panthera tigris	80.35
骆驼Camelus dromedarius	76.47	双峰驼Camelus bactrianus	74.47
蓝鲸Balaenoptera musculus	71.44	人Homo sapiens	68.23
狗Canis lupus familiaris	64.19	羊驼Vicugna pacos	62.73
鸭嘴兽Ornithorhynchus anatinus	62.44	大熊猫Ailuropoda melanoleuca	61.76
家鼠Mus musculus	53.87	鸡Gallus gallus	42.02
斑马鱼Danio rerio	38.34

SNP位点SNP site	基因型频率(样本数) Genotype frequency (sample number)	等位基因频率Allele frequency	Ho	He	Ne	PIC	χ² (HWE)
SNP1 (g.30667970T＞C)	TT：0.365(73)	T：0.575	0.511	0.489	1.956	0.369	3.957 (P＞0.05)
	TC：0.420(84)	C：0.425
	CC：0.215(43)
SNP2 (g.30668540T＞C)	GG：0.505(101)	G：0.723	0.599	0.401	1.669	0.321	1.439 (P＞0.05)
	TG：0.435(87)	T：0.278
	TT：0.060(12)
SNP3 (g.30668664C＞T)	CC：0.800(160)	C：0.900	0.820	0.180	1.220	0.164	2.469 (P＞0.05)
	CT：0.200(40)	T：0.100
	TT：0.000(0)

SNP位点SNP site	LD系数(D’) LD coefficient	相关系数(r²) Correlation coefficient
SNP1/SNP2	0.942	0.252
SNP1/SNP3	1.000	0.150
SNP2/SNP3	0.910	0.035

Polymorphism Analysis of the ATG16L2 Gene Promoter Region in Hainan Black Goat

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单倍型名称Haplotype name	单倍型Haplotype	频率F Frequency F
H1	CGC	0.321
H2	TGC	0.304
H3	TTC	0.271
H4	CGT	0.097

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