牛卵泡TEDDM1表达特点及其功能分析

Expression Characteristic and Function Analysis of TEDDM1 in Bovine Follicle

旨在研究牛卵泡跨膜附睾蛋白1(transmembrane epididymal protein 1,TEDDM1)的分子特征和立体结构,并结合TEDDM1在不同生理状态牛卵泡的表达特性分析其功能。本研究采集牛发情期第一卵泡波优势卵泡(dominant follicle,DF)和从属卵泡(subordinate follicle,SF),分别分离颗粒细胞(granulosa cells,GCs),提取总RNA后反转录,设计牛 TEDDM 1特异性引物进行PCR扩增、克隆、测序,获得全CDS区后运用生物信息学方法对序列结构进行分析;以牛 RPLP 0作为内参基因,在DF和SF中使用qRT-PCR对 TEDDM 1的表达量进行检测;兔抗TEDDM1一抗检测TEDDM1在牛卵泡中的表达和定位。结果表明, TEDDM 1基因CDS区全长903 bp,编码300个氨基酸,有7次跨膜的α螺旋结构,属于典型的G蛋白偶联受体,该基因氨基酸序列与非洲野牛( Bison bison bison )序列相似性最高,为99.4%;功能域分析表明,TEDDM1分子中存在未知功能结构域,蛋白家族716(domains of unknown function protein families 716,DUF716)结构域。qRT-PCR分析表明, TEDDM 1 mRNA在SF的表达量显著高于DF( P <0.05);免疫组化分析表明,TEDDM1在DF和SF的颗粒层和膜层细胞均有表达,特异性显色强度表明TEDDM1在SF颗粒层和膜层细胞表达量均高于DF。本研究为进一步探讨TEDDM1在牛卵泡发育过程中的调控作用及其在信号转导和激素调节中的功能提供了依据,为后期深入研究牛卵泡发育机理奠定基础。

The aim of this study was to identify molecular characteristic and three-dimensional structure of TEDDM1 in bovine follicle,its function was analyzed by combining expression characteristic of TEDDM1 in different physiology states of follicles. DF and SF in first follicular development wave during estrous cycle were collected,GCs were isolated,total RNA was extracted and the reverse transcription was performed by RT-PCR,the specific primers were designed to amplify TEDDM 1 and the sequence was sequenced,bioinformatics method was used to analyze CDS region sequence structure;Using RPLP 0 as reference gene,qRT-PCR was performed to detect the experssion of TEDDM 1 in DF and SF;The rabbit anti-TEDDM1 antibody was used to dectet expression level and localization of TEDDM1 in bovine follicles. The results showed that full length of TEDDM 1 CDS region was 903 bp,encoding 300 amino acids,contained 7 parallel alpha helical structures across the cell membrane,which was a typical G protein-coupled receptor,sequence of TEDDM 1 had the highest similarity with Bison (99.4%);The functional domain analysis showed that DUF716 existed in TEDDM1. The results of qRT-PCR analysis showed that expression level of TEDDM 1 mRNA in SF was significantly higher than that in DF ( P <0.05);The immunohistochemical analysis showed that TEDDM1 was expressed in GCs and membrane cells layer of DF and SF,and specific color intensity result showed that expression of TEDDM1 in SF GCs and membrane cells was higher than that in DF. The study provides a basis for further study on regulatory effect of TEDDM1 in bovine follicular development,signal transduction and hormone regulation,and lays a foundation for further research on mechanism of bovine follicular development.