摘要
以184头中国荷斯坦奶牛为试验对象,经PCR扩增、测序对比筛选单核苷酸多态性(single nucleotide polymorphisms,SNPs)位点,并与泌乳性状进行关联分型,进一步进行连锁不平衡及单倍型分析;生物信息学筛选靶向3′UTR区域SNPs位点的候选miRNAs,并预测候选miRNAs与该位点突变形成的2种基因型的mRNA靶向序列结合的最小自由能及二级结构,检测胰岛素样生长因子1(insulin-like growth factor 1,IGF-1)mRNA及蛋白质表达量,评估miRNA对牛乳腺上皮细胞脂滴数量及甘油三酯的含量的影响,进一步检测泌乳相关基因mRNA表达量,双荧光素酶报告试验探究miR-325-3p与IGF-1基因靶向关系及对不同基因型3′UTR沉默效率。结果显示,中国荷斯坦奶牛群体中共鉴定出7个IGF-1基因SNP位点,其中位于3′UTR的g.58240 G>A(c.756 G>A)对乳脂率和产奶量存在显著影响;生物信息学筛选到2种候选miRNAs(miR-325-3p及Y-74),其中miR-325-3p与靶向mRNA序列结合更加稳定;miR-325-3p-mimics组中ACSS1基因mRNA水平表达量较miR-325-3p-NC组显著下降(P<0.05),并且细胞内脂滴数量及甘油三酯含量均降低;miR-325-3p能够通过靶向IGF-1基因3′UTR,并且miR-325-3p对IGF-1基因沉默效率可能受IGF-1 c.756 G>A影响,进而影响IGF-1表达。本试验为解析IGF-1基因对泌乳性状的影响,以及IGF-1基因对牛分子育种的作用奠定了基础。
A total of 184 Chinese Holstein dairy cows were selected as the research subjects.The single nucleotide polymorphisms(SNPs)were screened by PCR amplification and sequencing.The association between SNPs and lactation traits was typed,and the linkage disequilibrium and haplotype analysis were further performed.Bioinformatics was used to screen candidate miRNAs targeting SNPs in the 3'UTR region,the minimum free energy and secondary structure of candidate miRNAs binding to the mRNA targeting sequences of the two genotypes formed by mutations in this site were predicted.Insulin-like growth factor 1(IGF-1)mRNA and protein expression levels were detected.The effects of miRNA on the lipid droplets number and the content of triglyceride in bovine mammary epithelial cells were evaluated,and the mRNA expression of lactation-related genes was further detected.The dual luciferase reporter assay was used to explore the targeting relationship between miR-325-3p and IGF-1 gene and the silencing efficiency of 3'UTR in different genotypes.The results showed that a total of 7 SNPs of IGF-1 gene were identified in the Chinese Holstein dairy cattle population,and the g.58240 G>A(c.756 G>A)located in the 3'UTR had significant effects on milk fat percentage and milk yield.Two candidate miRNAs(miR-325-3p and Y-74)were screened by bioinformatics,and miR-325-3p was more stable in binding to the target mRNA sequence.The expression of ACsS1 mRNA in the miR-325-3p-mimics group was significantly lower than that in the miR-325-3p-NC group(P<0.05),and the number of lipid droplets and the content of TAG in the cells were decreased.miR-325-3p can target the 3'UTR of IGF-1gene,and the silencing efficiency of miR-325-3p on IGF-1 gene may be affected by IGF-1 c.756 G>A,thereby affecting the expression of IGF-1.This study laid the foundation for analyzing the effect of IGF-1 gene on lactation traits and the role of IGF-1 gene in cattle molecular breeding.
作者
房嘉园
张丽波
郑硕
吕秦川
苏桐
张洵铭
肖兴玉
李一
李常红
郝林琳
FANG Jiayuan;ZHANG Libo;ZHENG Shuo;LYU Qinchuan;SU Tong;ZHANG Xunming;XIAO Xingyu;LI Yi;LI Changhong;HAO Linlin(College of Animal Science,Jilin University,Changchun 13o062,China;College of Life Science,Baicheng Normal University,Baicheng,Jilin 137000,China)
出处
《中国兽医学报》
2026年第1期156-165,174,共11页
Chinese Journal of Veterinary Science
基金
吉林省科学技术厅科技发展计划资助项目(20230202068NC)。
