香猪CHD3基因3′-侧翼区264 bp结构变异的研究

Study on 264 bp Structural Variation of 3′-flanking Region of CHD3 Gene in Xiang Pigs

【目的】探究染色质调节域解旋酶DNA结合蛋白3(chromodomain-helicase-DNA-binding protein 3,CHD3)基因3′-侧翼区的一段264 bp核苷酸结构变异对CHD3基因表达的影响,并初步解析该结构变异与香猪皱皮形成的关系。【方法】以普通香猪、皱皮香猪、大白猪为研究对象,应用UCSC、miRBase、miRanda、RBPsuite软件预测CHD3基因结构变异区间所包含的重复元件、miRNA结合位点、RNA结合蛋白(RBP)的结合位点;采用PCR方法对结构变异位点进行基因分型;利用Excel完成群体基因型频率和等位基因频率的计算,同时χ^(2)检验分析基因型是否处于Hardy-Weinberg平衡状态;用实时荧光定量PCR和Western blotting检测该变异对CHD3基因表达的影响。【结果】生物信息学分析显示,CHD3基因3′-侧翼区的结构变异是1个长254 bp的短散在元件(SINE),属于tRNA家族,位于Chr12:53115742-53115995,该片段含有35个miRNA结合位点和10个RBP结合位点。基因分型显示,在皱皮香猪、普通香猪、大白猪3个猪群中均检测到了II基因型(插入型)、ID基因型(杂合型)、DD基因型(双缺失),表明CHD3基因3′-侧翼区的264 bp结构变异在猪群中呈现丰富的多态性。基因型群体分布频率显示,皱皮香猪中的D等位基因频率极显著高于普通香猪和大白猪(P<0.01),该结构变异在皱皮香猪和普通香猪中处于Hardy-Weinberg平衡状态(P>0.05),在大白猪中处于Hardy-Weinberg不平衡状态(P<0.05)。实时荧光定量PCR和Western blotting检测显示,香猪皮肤中CHD3基因的DD基因型和ID基因型mRNA和蛋白表达量均极显著高于II基因型(P<0.01)。【结论】CHD3基因3′-侧翼区的264 bp结构变异是1个具有调控作用的SINE元件,通过RNA结合蛋白和miRNA与该元件的结合,促进CHD3基因mRNA的代谢和转录后的翻译抑制,从而调节CHD3基因的表达,结构变异导致CHD3基因的异常表达和积累,可能与香猪皱皮形成有关。

【Objective】This study was aimed to explore the effect of structural variation of chromodomain-helicase-DNA-binding protein 3(CHD3)gene on the expression of CHD3 gene in different pig breeds,and preliminarily analyze the relationship between the CHD3 gene structural variation and the formation of wrinkle skin in the Xiang pigs.【Method】In this study,ordinary Xiang pigs,Xiang pigs with wrinkle skin and Large White pigs were selected as the research subjects.UCSC,miRBase,miRanda and RBPsuite softwares were used to predict the repeated elements,miRNA binding sites and RNA binding protein(RBP)binding sites contained in the CHD3 structural variation interval.PCR method was used for genotyping of structural variation.Excel was used to calculate the population genotype frequency and allele frequency,andχ^(2)test was used to analyze whether the genotype was in Hardy-Weinberg equilibrium.The effect of the variation on CHD3 gene expression was detected by Real-time quantitative PCR and Western blotting.【Result】Bioinformatics analysis showed that the 3′-flanking region of CHD3 gene was a 254 bp short-scatter element(SINE),belonging to tRNA family,located in Chr12:53115742-53115995,which contained 35 miRNA binding sites and 10 RBP binding sites.Genotyping results showed that II genotype(insertion),ID genotype(heterozygous)and DD genotype(double deletion)were detected in all three pig groups,indicating that 264 bp structural variation in the 3′-flanking region of CHD3 gene was highly polymorphic in pigs.Genotype population distribution frequency showed that the frequency of D allele in Xiang pigs with wrinkle skin was extremely significantly higher than that in ordinary Xiang pigs and Large White pigs(P<0.01);χ^(2)test showed that the structural variation of CHD3 gene was in Hardy-Weinberg equilibrium state in Xiang pigs with wrinkle skin and ordinary Xiang pigs(P>0.05),but unbalanced in Large White pigs(P<0.05).Real-time quantitative PCR and Western blotting analysis showed that the mRNA and protein expression of DD and ID genotypes of CHD3 in skin of Xiang pigs were extremely significantly higher than that of II genotype(P<0.01).【Conclusion】The 264 bp structural variation in the 3′-flanking region of CHD3 gene was a SINE element with regulatory effect,which could promote the metabolism of CHD3 gene mRNA and post-transcriptional translation inhibition through the binding of RNA binding protein and miRNAs to the element,thus regulating the expression of CHD3 gene.The structural variation resulted in abnormal expression and accumulation of CHD3 gene,which might be related to the formation of wrinkled skin in Xiang pigs.