Effects of Genetic Variation of MIR206 on Growth Traits and Promoter Activity of Anhui White Goat (Capra hircus)
DUAN Qin1, DING Yang1, LU Jia-Ni1, ZHANG Si-Huan1,*, LING Ying-Hui1,2,*
1 College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China; 2 Local Livestock Genetic Resource Conservation and Breeding Key Laboratory of Anhui Province, Hefei 230036, China
摘要MIR206是MIR-1家族成员之一,在脊椎动物骨骼肌中特异表达,而肌肉是机体的重要组成部分,因此MIR206基因可能对动物的生长发育具有重要调控作用。插入/缺失(Insertion/Deletion, InDel)是一种重要的遗传变异,可能通过影响基因表达水平影响基因功能。本研究探究了安徽白山羊(Capra hircus) MIR206基因InDel变异对生长性状及启动子活性的影响,以期为山羊分子育种提供参考。以431只安徽白山羊为研究对象,收集相应个体的体重、体高、体长、胸围、管围和尻宽等数据。采用PCR产物直接测序的方法鉴定MIR206基因的InDel突变。利用PCR和琼脂糖凝胶电泳鉴定安徽白山羊个体的基因型;通过计算遗传参数分析InDel变异在安徽白山羊群体中的分布情况;利用SPSS 25.0分析突变位点基因型与山羊生长性状的相关性;通过双荧光素酶报告系统检测分析MIR206启动子区遗传变异对启动子活性的影响。PCR产物测序和序列比对结果显示,安徽白山羊MIR206转录起始位点上游389~394位置存在1个6 bp InDel变异(rs653221349)。该变异在安徽白山羊群体中有3种基因型：II (野生型)、ID (杂合型)和DD (纯合缺失型),属于中度多态(0.25<PIC<0.5),处于Hardy-Weinberg不平衡状态(P<0.05)。关联分析结果显示,6 bp InDel的不同基因型与安徽白山羊的体重、体长和胸围显著相关(P<0.05)。其中ID基因型个体的体重、体长和胸围显著优于II和DD基因型个体。双荧光素酶报告系统结果显示,6 bp的缺失显著降低了基因启动子活性。综上,本研究发现安徽白山羊MIR206基因动子区存在6 bp InDel变异,该突变与安徽白山羊的部分生长性状显著相关且降低了启动子活性,可作为安徽白山羊分子标记辅助选择的候选DNA分子标记。
Abstract：MIR206 is a member of the MIR-1 family, which specifically expressed in skeletal muscle of vertebrates. Skeletal muscle is an important part of the body, so MIR206 gene may play an important role in the regulation of animal growth and development. Insertion/Deletion (InDel) is a kind of important genetic variation that may affect gene function by regulating gene expression levels. Therefore, this study investigated the effects of MIR206 InDel variation on growth traits and MIR206 promoter activity of Anhui white goat (Capra hircus) in order to provide theoretical reference for molecular breeding of goat. The body weight, body height, body length, chest circumference, cannon circumference, and rump width data of 431 Anhui white goat were collected. The InDel mutations of MIR206 gene were identified by direct sequencing of PCR products. The genotypes of Anhui white goat were detected by PCR and agarose gel electrophoresis. The distribution of InDel variation in Anhui white goat population was analyzed by calculating genetic parameters. SPSS 25.0 was used to analyze the association between the genotypes and the growth traits of goats. The effect of the genetic variation in the promoter region of MIR206 on promoter activity was analyzed by dual luciferase reporting system. The results of PCR product sequencing and sequence alignment showed that there was a 6 bp InDel mutation (rs653221349) at the 389~394 locus in front of the transcriptional start site of MIR206. There were 3 genotypes of this variant in Anhui white goat population: II (wild genotype), ID (heterozygous genotype) and DD (homozygous deletion genotype), which belonged to moderate polymorphism (0.25<PIC<0.5) and was in Hardy-Weinberg imbalance (P<0.05). The results of association analysis showed that different genotypes of the 6 bp InDel were significantly correlated with body weight, body length and chest circumference of Anhui white goat (P<0.05). The body weight, body length and chest circumference of ID genotype individuals were significantly better than those of II and DD genotype individuals. Results of the dual luciferase reporting system showed that the 6 bp deletion significantly reduced gene promoter activity. In summary, this study found that there was a 6 bp InDel mutation in the promoter region of MIR206 gene of Anhui white goat, which was significantly associated with growth traits of Anhui white goat and reduced promoter activity, which could be used as a candidate DNA molecular marker for molecular marker-assisted selection of Anhui white goat.
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