绵羊清道夫受体A基因启动子克隆及序列分析

Cloning and sequence analysis of sheep scavenger receptor A gene promoter

为了分析绵羊清道夫受体A(scavenger receptor A,SRA)基因的启动子序列特征,试验根据UCSC数据库和NCBI数据库预测的绵羊SRA基因启动子序列设计特异性引物,以绵羊肺组织基因组DNA为模板进行SRA基因启动子序列PCR扩增、测序,然后对序列进行生物信息学分析,包括预测启动子活性区域、转录因子结合位点、TATA box及CpG岛。结果表明:利用根据UCSC数据库预测的SRA基因启动子序列设计的引物未扩增出目的片段,利用NCBI数据库预测的SRA基因启动子序列设计的引物扩增得到了绵羊SRA基因启动子序列,大小约为1200 bp,与NCBI预测序列的相似性为99%,其中第577位碱基发生了突变(G→A);含有1个潜在活性区域(aaaaatgagctcacattcattttttttttcttaactggc);存在干扰素调节因子(interferon regulatory factor,IRF)1、IRF2、c-Jun、CCAAT增强子结合蛋白(CCAAT enhancer binding protein,C/EBP)、活化蛋白1(activator protein 1,AP-1)、核因子κB(nuclear factor kappa-B,NF-κB)转录因子结合位点,其中IRF1和c-Jun的结合位点出现频率较高;不存在TATA box和CpG岛,只存在3个CpG位点。说明SRA基因的表达可能受IRF1和c-Jun等相关转录因子的调控,不受甲基化的影响。

In order to analyze the promoter sequence characteristics of sheep scavenger receptor A(SRA)gene,specific primers were designed based on the predicted sheep SRA gene promoter sequence from the UCSC database and the NCBI database,and sheep lung tissue genomic DNA was used as a template for PCR amplification and sequencing of the SRA gene promoter sequence,and then bioinformatics analysis was performed on the sequence,including the prediction of the promoter active region and binding site of transcription factors and CpG islands.The results showed that the target fragment was not amplified by using primers designed based on the predicted SRA gene promoter sequence from the UCSC database,and the sheep SRA gene promoter sequence was amplified by using primers designed based on the predicted SRA gene promoter sequence from the NCBI database;the size was about 1200 bp,and its similarity with the predicted NCBI sequence was 99%,in which a base mutation(G→A)occurred at nucleotide 577.The sequence contained one potential active region(aaaaatgagctcacattcattttttttttcttaactggc)and binding sites of interferon regulatory factor 1(IRF-1),IRF-2,c-Jun,CCAAT enhancer binding protein(C/EBP)and nuclear factor kappa-B(NF-κB)transcription factor,among which the frequency of IRF1 and c-Jun binding sites was higher.But there was no TATA boxes and CpG islands,and only three CpG sites presented.The results indicated that the expression of SRA gene might be regulated by IRF1 and c-Jun and not affected by methylation.