山羊PGRMC1基因序列分析及表达特性分析

Analysis of PGRMC1 gene and its expression characteristics in goats

旨在克隆获得山羊PGRMC1基因序列,并明确该基因的生物学特征,阐明其在山羊各组织及诱导分化不同阶段皮下脂肪细胞中的表达模式.利用RT-PCR技术克隆获得山羊PGRMC1基因序列,通过生物信息学方法分析其生物学特性;通过实时荧光定量PCR(Real-time fluorescence quantitative PCR,RT-qPCR)技术检测其在各个组织及不同分化阶段皮下脂肪细胞中的表达水平.结果显示,克隆得到山羊PGRMC1基因序列1 024 bp,其中CDS区585 bp,共编码194个氨基酸,是主要定位于细胞质、细胞核以及线粒体的酸性亲水性不稳定蛋白;分析显示山羊PGRMC1的核苷酸序列与其他物种如绵羊、牛、猪、羊驼、猩猩、人、马、猫、骆驼、狐狸相似程度为88.79%~99.69%不等,说明该基因在不同物种中具有高度保守性;构建进化树显示,山羊PGRMC1与绵羊亲缘关系最近,与马的亲缘关系最远,符合物种进化规律;组织表达谱显示,PGRMC1在山羊心、肝、脾、肺、肾、背最长肌中广泛表达,其中在肝脏中的表达量极显著高于其他组织(P<0.01),其次是在背最长肌和脾脏中的表达水平较高且显著高于肝脏之外的组织(P<0.05);时序表达谱显示,山羊PGRMC1基因在成脂诱导分化96 h的皮下脂肪细胞中的表达量最高,显著高于其他各个时间点的表达水平(P<0.05).本研究将为揭示山羊PGRMC1调控皮下脂肪细胞分化的具体作用机制提供理论依据.

This study aimed to clone the PGRMC1 gene sequence of goats and clarify its biological characteristics.The expression pattern of PGRMC1 in subcutaneous adipocytes in various tissues of goats and induced differentiation at different differentiation stages waselucidated.In this study,the PGRMC1 gene sequence of goats was cloned by RT-PCR,and its sequence characteristics were analyzed by bioinformatics analysis software,and RT-qPCR(Real-time fluorescence quantitative PCR)was used to detect its expression in various tissues and in subcutaneous adipocytes at different stages of differentiation.The results showed that the gene sequence of PGRMC1 in goats was 1024 bp,of which 585 bp in the CDS region,encoded a total of 194 amino acids,and was an acidic hydrophilic unstable protein mainly localized in the cytoplasm,nucleus and mitochondria.The nucleotide sequence of PGRMC1 in goats ranged from 88.79%to 99.69%in similarity with other species such as sheep,cattle,pigs,alpacas,orangutans,humans,horses,cats,camels,and foxes,indicating that PGRMC1 was highly conserved in different species.The results of phylogenetic tree analysis showed that goat PGRMC1 was most closestly related to sheep and most distantly related to horses,which was consistent with the evolutionary law of species.Tissue expression profiles showed that PGRMC1 was widely expressed in goat heart,liver,spleen,lung,kidney,and longest dorsal muscle,among which the expression in the liver was significantly higher than that of other tissues(P0.01),followed by higher expression levels in the longest dorsi muscle and spleen than in tissues other than the liver(P 0.05).Compared with the control group,the expression of PGRMC1 during subcutaneous adipocyte differentiation showed an upward trend,and reached the highest at 96 h,which was significantly higher than other time points.The above results suggested that PGRMC1 might have an important regulatory effect on the differentiation of goat adipocytes.