【摘要】 【目的】明确泛素特异性蛋白酶18基因(USP18)在猪不同妊娠时期子宫内膜中的表达规律及其与雌激素和孕激素的关系,并分析其多态性与产仔数性状的关联性,为揭示USP18基因在猪妊娠过程中的作用机理提供参考依据。【方法】PCR扩增猪USP18基因编码区(CDS)序列及开展相关生物信息学分析,利用实时荧光定量PCR检测USP18基因在梅山猪和大白猪妊娠15、26和50 d子宫内膜中的表达水平,通过体外细胞试验检测分析雌二醇和孕酮联合处理对子宫内膜上皮细胞USP18基因表达的影响,并采用PCR-RFLP和SPSS 19.0检测猪USP18基因多态性与产仔数性状的关联性。【结果】猪USP18基因CDS序列为966 bp,共编码322个氨基酸残基,编码蛋白分子量为37 kD,理论等电点(pI)为6.74;猪USP18氨基酸序列与牛USP18氨基酸序列的同源性为82%,二者的遗传距离最近。USP18基因在妊娠15和26 d梅山猪和大白猪子宫内膜中的相对表达量均明显高于妊娠50 d,且在妊娠26和50 d,USP18基因在梅山猪子宫内膜组织中的相对表达量显著高于在大白猪子宫内膜组织中的相对表达量(P<0.05,下同)。以10-10mol/L雌二醇和10-8mol/L孕酮联合处理子宫内膜上皮细胞,USP18基因在子宫内膜上皮细胞中的相对表达量呈上调趋势,且显著高于激素处理前的相对表达量。在猪USP18基因CDS序列953 bp处存在1个SNP位点(T/G错义突变),其不同基因型与产仔数性状的关联分析结果表明,在经产母猪中,TG基因型母猪产活仔数最多,GG基因型母猪产活仔数最少,且差异显著。【结论】猪USP18基因在附植期子宫内膜中高水平表达,且在子宫内膜上皮细胞中的表达受雌激素和孕激素诱导,表明USP18基因在妊娠早期建立过程中发挥着重要作用。USP18基因在梅山猪和大白猪间的差异表达可能是导致梅山猪产仔数高于大白猪的一个重要因素。更多还原

【Abstract】 【Objective】The aim of study was to clarify the expression of porcine ubiquitin specific peptidase 18 gene(USP18)in Meishan pig and Yorkshire pig endometrium during different gestation stages,examine the relationship between its expression and estrogen and progesterone,analyze the association between USP18 gene polymorphism and li-tter size,and thus to provide important data for revealing the role of USP18 gene in the process of pregnancy.【Method】The PCR was used to amplify protein coding region(CDS)of USP18 gene,and bioinformatics analysis was conducted.Real-time fluorescence quantitative PCR was used to analyze the USP18 gene expression level in endometrium of Meishan pig and Yorkshire pig on 15 d,26 d and 50 d of pregnancy. The cell transfection technology was used to detected the effect of estradiol and progesterone on the USP18 gene expression in endometrial epithelial cells. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)and SPSS 19.0 software were used to analyze the association between the gene polymorphism of USP18 and reproductive traits.【Result】The CDS sequence of pig USP18 gene was 966 bp. It encoded 322 amino acids,and the molecular weight and the isoelectric point(pI)were 37 kD and 6.74 respectively. Amino acid sequence of USP18 protein had 82% similarity with cattle,and their genetic distance was the nearest. The expression level of USP18 gene on day 15 and day 26 of pregnancy were higher compared with day 50 of pregnancy both in Meishan and Yorkshire endometriums,and the expression in Meishan pig endometrium was significantly higher than in Yorkshire pig endometriums both on day 26 and day 50 of pregnancy(P<0.05,the same below). Moreover,treating with 10-10 mol/L estradiol and 10-8 mol/L progesterone in endometrial epithelial cells,the expression of USP18 was induced and was significantly higher than that before hormone treatment. Gene polymorphism analysis indicated that USP18 CDS sequence had a SNP site(T/G missense mutation)at 953 bp. The association analysis between various genotypes and litter size traits indicated that among multiparous sows,TG genotpye sows had the largest litter size and GG genotpye sows had the smallest litter size,and with significant difference.【Conclusion】USP18 gene has high level expression in endometriums during implantation and is induced by estrogen and progesterone. This results indicates that USP18 may play important roles in the establishment of early pregnancy. And the differential expression in endometrium between Meishan pig and Yorkshire pig may be an important reason which leads to the litter size of Meishan pig higher than Yorkshire pig.更多还原