比格犬雌激素受体β四个剪接异构体的发现

Discovery of four splicing isoforms of Beagle estrogen receptor β

目的在比格犬下丘脑-垂体-性腺轴上筛查雌激素受体β的剪接异构体。方法针对ERβmRNA CDS序列的八个外显子,两两外显子依次组合设计引物,运用PCR法,以比格犬下丘脑、垂体、卵巢、子宫组织c DNA为模板扩增相应序列,测序后在NCBI网站比对,确定是目的基因后,用DNAMAN软件比对分析及手工校对,获得ERβ的剪接异构体。结果获得到了比格犬ERβ的四个剪接异构体,分别是第4外显子完整缺失的ERβ剪接异构体(缺失300 bp),部分第4与部分第5外显子组合缺失的两个组合型缺失的ERβ剪接异构体(各缺失334bp,265 bp),以及第七外显子完整缺失的ERβ剪接异构体(缺失181 bp)。第4外显子完整缺失的剪接异构体和第7外显子完整缺失的剪接异构体获得了全长编码序列,另两个剪接异构体为部分编码序列。结论研究获得了比格犬ERβ的四个剪接异构体,为后续研究其在比格犬生殖调控中的作用机制打下了基础。

Objective To screen the splicing isoforms of estrogen receptor β in the Beagle hypothalamic - pituitary - gonadal axis. Methods For ERβ mRNA CDS sequence of eight exons, primers were designed confined to the CDS sequences of two sequential exons. Beagle hypothalamus, pituitary, ovary and uterus tissue cDNA were used as template, and corresponding sequences were amplified by PCR. PCR products were sequenced and aligned in the NCBI web site. The correct gene was then analyzed with DNAMAN comparative analysis software and handwork checking up, thus got the ERβ splicing isoforms of Beagle. Results Four beagle ER beta splicing isomers were obtained： exon 4 complete skipping ERβ isomer （300 bp missing）, two kind of Beagle ERβ isoforms with partial exon 4 and partial exon 5 complicated missing （isoform I334 bp missing and isoform Ⅱ 265 bp missing）, and exon 7 complete missing ERβ splicing isoforms （ 181 bp missing）. Exon 4 complete skipping and exon 7 complete missing isomers had been obtained full length coding sequence, and the other two splicing isomers were partial coding sequence. Conclusion This project gained four ER β splicing isomers of Beagle, and that will lay an important foundation for further study of their roles in the Beagle reproductive regulation mechanism.