香猪CYP3A29基因3′-UTR结构变异及其与肌肉雄激素水平的关联分析

Association Analysis Between Structural Variation of CYP 3 A 29 Gene 3′-UTR and Androgen Level of Muscle Tissue in Xiang Pigs

【目的】探究猪特有的细胞色素P450超家族3A亚族成员29(cytochrome P4503A 29,CYP3A29)基因3′-UTR区核苷酸结构变异(structural variation,SV)对该基因表达的影响,解析该结构变异与香猪肉质性状形成的关系。【方法】以香猪、大白猪为研究对象,应用UCSC、miRBase、PITA、RBPsuite等软件预测CYP 3 A 29基因3′-UTR区结构变异区间所包含的重复元件、miRNA和RBP结合位点;采用PCR方法对结构变异位点进行基因分型;计算群体基因型频率、基因频率及Hardy-Weinberg平衡状态;采用实时荧光定量PCR检测该变异对CYP 3 A 29基因表达的影响;利用ELISA法检测香猪肌肉组织中CYP3A29蛋白含量和雄激素(T、DHT)水平,并使用SPSS 17.0软件对T、DHT含量与不同基因型进行Pearson相关性分析。【结果】生物信息学分析显示,CYP 3 A 29基因3′-UTR的结构变异区全长303 bp,该变异区存在1个长为241 bp的短散在元件(short interspersed element,SINE),属于Pre0_SStRNA家族,位于Chr3:6819917―6820161,含有33个miRNAs和13个RBPs结合位点。基因分型显示,该结构变异在香猪、大白猪群体中呈现出丰富的多态性,表现为II基因型(插入型)、ID基因型(杂合型)、DD基因型(双缺失)。基因型群体分布频率显示,香猪中的D等位基因频率高于大白猪,该结构变异在香猪中处于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ELISA结果显示,CYP 3 A 29基因ID基因型个体mRNA表达量和蛋白含量均显著高于II和DD基因型(P<0.05)。相关性分析结果显示,香猪不同基因型个体CYP3A29基因含量与肌肉组织中T浓度呈显著相关(P<0.05),与DHT浓度不相关(P>0.05)。香猪ID基因型个体T浓度显著高于DD和II基因型(P<0.05),DD基因型个体DHT浓度显著高于ID和II基因型(P<0.05)。【结论】CYP 3 A 29基因3′-UTR区长度为303 bp的结构变异在香猪群体中具有多态性,以缺失型D等位基因为主,即该结构变异在香猪中缺失SINE元件,导致CYP 3 A 29基因表达上调,进而影响香猪肌肉组织中T浓度。CYP 3 A 29基因3′-UTR结构变异与香猪肉质性状具有相关性。

【Objective】This study was conducted to investigate the effect of the structural variation(SV)in the pig-specific cytochrome P4503A 29(CYP3A29)gene 3′-UTR on its expression,and analyze the relationship between the SV and the formation of meat quality traits in Xiang pigs.【Method】The repeated elements,miRNA,and RBPs binding sites in the SV of CYP 3 A 29 gene 3′-UTR were predicted by UCSC,miRBase,PITA and RBPsuite softwares in Xiang pigs and Large White pigs.The SV site was genotyped by PCR,the genotype frequency,gene frequency and Hardy-Weinberg equilibrium were calculated.The mRNA expression of CYP 3 A 29 gene was detected by Real-time quantitative PCR.The content of CYP3A29 protein and the level of androgen(T and DHT)in muscle were detected by ELISA.Pearson correlation analysis between the contents of T and DHT and different genotypes was performed by SPSS 17.0 software.【Result】Bioinformatics analysis results showed that the SV length of CYP 3 A 29 gene 3′-UTR was 303 bp,and there was a short interspersed element(SINE)with a length of 241 bp in this variation,belonging to Pre0_SStRNA family,located in Chr3:6819917―6820161,containing 33 miRNAs and 13 RBPs binding sites.The genotyping confirmed that the SV in Xiang pigs and Large White pigs showed rich polymorphism,which contained insertion(II),hybrid(ID)and deletion(DD).The frequency of D allele in Xiang pigs was higher than that in Large White pigs.The SV was in Hardy-Weinberg equilibrium in Xiang pigs(P>0.05),but not in Large White pigs(P<0.05).The results of Real-time quantitative PCR and ELISA showed that the mRNA expression and protein content of ID genotype of CYP 3 A 29 gene in Xiang pigs were significantly higher than II and DD genotypes,respectively(P<0.05).The correlation analysis results showed that the expression of CYP3A29 gene of different genotypes was significantly correlated with T concentration of muscle tissue in Xiang pigs(P<0.05),but not correlated with DHT concentration(P>0.05).The T concentration of ID genotype in Xiang pigs was significantly higher than that of DD and II genotypes(P<0.05),and DHT concentration of DD genotype was significantly higher than that of ID and II genotypes(P<0.05).【Conclusion】The SV of 303 bp in CYP 3 A 29 gene 3′-UTR was polymorphic in Xiang pigs,and the deletion D allele was the main one,that was the SV lacked the SINE element in Xiang pigs,which leaded to the up-regulation of CYP 3 A 29 gene expression,thereby affecting the T concentration of muscle tissue in Xiang pigs.In summary,the SV of CYP 3 A 29 gene 3′-UTR was correlated with the meat quality traits in Xiang pigs.