鸡GNAS基因c.36-2277T>C突变对萤光素酶表达的影响

Effect of c.36-2277T>C mutation on the expression of luciferase in chicken GNAS gene

为分析探讨鸡GNAS基因ENSGALT00000062075.1:c.36-2277T>C突变对转录调控的影响,本试验选择乌骨鸡和芦花鸡血液样本,构建了c.36-2277T>C突变点的不同基因型质粒,并转染鸡胚成纤维细胞后,检测双萤光素酶报告基因分析系统的表达。结果表明:样本芦花鸡均为TT基因型,乌骨鸡均为CC基因型;c.36-2277T>C突变点的上下游-175～+818 bp片段具有显著增强转录活性作用(P<0.01),且CC基因型活性极显著高于TT基因型(P<0.000 1)。转录因子预测结果表明:此993 bp片段中TGGCA结合蛋白位点数量最多;紧邻突变点分别为AP-1和ER-alpha/T3R-alpha结合位点。研究认为,GNAS基因c.36-2277T>C突变点的碱基类型会对基因转录调控进程产生重要影响,且该突变可能是通过与上下游转录因子结合位点共同作用来发挥效用。

In order to analyze the effect of ENSGALT00000062075.1:c.36-2277T > C mutation on the expression of transcriptional regulation in chicken GNAS gene, this study collected blood DNA samples from black-bone chicken and barred chicken to construct different genotypic plasmids of c. 36-2277T>C mutation, and detected the expression of dual-luciferase reporter analysis system after transfecting chicken embryo fibroblasts. The result showed that all barred chicken samples belonged to TT genotypes and all black-bone chicken samples were CC genotypes. The section from 175 bp upstream to 818 bp downstream of c.36-2277T> C mutation had significantly transcript enhancing activity (P<0.01). The activity of CC genotype was significantly higher than that of the TT genotype (P<0.000 1). The result of transcript factor prediction showed that the number of TGGCA-binding protein sites was the largest in this 993 bp section, and the adjacent mutations were AP-1 and ER-alpha / T3R-alpha binding sites. The study suggests that the base types of c. 36-2277T>C mutation in GNAS gene have an important impact on the process of gene transcription regulation, and this mutation plays the role associated with upstream and downstream binding sites of transcript factors.