海南黑山羊ATG16L2基因启动子区多态性研究

Polymorphism Analysis of the ATG 16L2 Gene Promoter Region in Hainan Black Goat

旨在研究海南黑山羊ATG16L2基因启动子区的结构特征及其遗传分布情况,为进一步探索该基因的表达调控机制及功能提供理论依据。本研究以200头海南黑山羊为研究对象,构建DNA混池,采用Sanger法测序对海南黑山羊ATG16L2基因启动子区的多态性进行初筛,应用PCR-RFLP技术对200头海南黑山羊个体进行基因型鉴定。对筛选到的SNP位点进行连锁不平衡分析,构建单倍型。利用生物信息学方法分析SNP位点对海南黑山羊ATG16L2基因表达的影响。在海南黑山羊ATG16L2基因启动子区共检测到3个SNPs位点,分别为SNP1(g.30667970T>C)、SNP2(g.30668540T>C)和SNP3(g.30668664C>T),且彼此连锁。SNP1和SNP2位点均表现为中度多态性,SNP3位点表现为低度多态性,且符合Hardy-Weinberg平衡(P>0.05)。单倍型分析结果显示,H1、H2、H3和H4单倍型频率分别为0.321、0.304、0.271和0.097,且H1(CGC)为优势单倍型。生物信息学分析显示,山羊ATG16L2基因共预测到3个启动子和4个CpG岛区域;存在2个重复元件LINE2(-1989~-1826 bp、-562~-426 bp)、hAT-Charlie(-1804~-1511 bp)以及5个CCAAT-Box、13个CAAT-Box、10个CGCG-Box、11个GATA-Box和2个TATA-Box。综合多种在线软件预测发现,上述SNPs可能通过影响ATG16L2基因的启动子区的顺式作用元件,从而影响海南黑山羊ATG16L2基因的转录表达。本研究在海南黑山羊ATG16L2基因启动子序列中发现3个SNPs位点,其中SNP1和SNP2表现为中度多态性,SNP3表现为低度多态性,并预测这些SNPs可能影响转录因子结合,从而调控基因表达,为进一步探究ATG16L2基因功能及其调控机制提供了理论依据。

The study aimed to investigate the structural characteristics and genetic distribution of the ATG 16L2 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 ATG 16L2 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 ATG 16L2 gene in Hainan Black goats.The promoter region of the ATG 16L2 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(-1989--1826 bp,-562--426 bp),hAT-Charlie(-1804--1511 bp),and 5 CCAAT-Box,13 CAAT-Box,10 CGCG-Box,11 GATA-Box and 2 TATA-Box of the ATG 16L2 gene in Hainan Black goat.The integrated prediction from various online tools suggests that the above SNPs may affect the expression regulation of the ATG 16L2 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 ATG 16L2 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 ATG 16L2 gene.