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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 Beagle dog hypothalamic-pituitary-gonad axis. Methods Eight exons and two exons of ERβmRNA CDS sequences were designed and synthesized in turn. PCR was used to amplify the corresponding sequences of c DNA of hypothalamus, pituitary, ovary and uterus in beagle dogs. After sequencing After aligning on the NCBI website and confirming the target gene, DNAMAN software was used for alignment analysis and manual proofreading to obtain the ERβ splicing isoforms. RESULTS: Four splice variants of ERβ were obtained, which were the ERβ splice variant (deletion of 300 bp) of the complete deletion of exon 4 and the deletion of two partial deletion of exon 4 and partial exon 5 ERβ splice variants (334bp and 265 bp each) with deletion of the combined ERβ splice variant (deletion of 181 bp) with the deletion of the seventh exon. The full-length coding sequence is obtained for the splicing isoform of the entire exon 4 deletion and the splice-isomer of the complete deletion of exon 7, and the other two splice isoforms are partial coding sequences. Conclusion The four splicing isoforms of ERβ in beagle dogs were obtained, which laid the foundation for further studies on their mechanism of action in reproductive regulation of beagle dogs.