水牛NOBOX基因启动子克隆及转录活性检测

Promoter cloning of buffalo NOBOX gene and detection of the transcriptional activity

为了对广西本地水牛新生儿卵巢同源盒基因(NOBOX基因)5'侧翼序列进行克隆、生物信息学分析及其转录活性进行研究,试验采用Gen Bank上已公布的黄牛NOBOX基因5'侧翼序列设计引物,以广西本地水牛血液基因组为模板扩增NOBOX基因5'侧翼序列并进行生物信息学分析。结果表明:研究成功克隆得到广西本地沼泽型水牛NOBOX基因5'侧翼序列及部分CDS区序列,共2 906 bp。同源性分析结果表明,其与地中海水牛、黄牛、绵羊和山羊的相似性分别为99%、96%、91%、91%。试验对NOBOX基因5'侧翼序列2 000 bp进行启动子及转录因子结合位点预测,在其翻译起始位点上游-868/-853 bp处存在TATA box,启动子区存在GATAs、Foxo1、Foxo3、Nobox、Stat1、Stat3、Stat4、Stat5a、Stat5b、Stat6、YY1等反式作用元件结合位点,其中GATAs家族基因在NOBOX启动子区存在多个结合位点,且同一位点又存在多个GATA基因结合。水牛NOBOX启动子能够启动EGFP在HEK-293T细胞上表达,但表达非常微弱,也能够启动EGFP在CHO细胞上表达,且与CMV启动EGFP在CHO细胞中的表达强度相似,表明水牛NOBOX是个强启动子。

To clone the 5'flanking sequence of the NOBOX gene in new- born local swamp buffalo from Guangxi,and make a bioinformatics analysis and study the transcription activity,primers were designed by the 5'flanking sequence of the cattle NOBOX gene published on Gen Bank.The genome in the blood of local swamp buffalo from Guangxi as a temple was used to amplify the 5'flanking sequence of the NOBOX gene,and then a bioinformatics analysis was conducted in the test. The results showed the 5 'flanking sequence of the NOBOX gene in local swamp buffalo from Guangxi partial sequence from the CDS region were successfully cloned,and the sizes of these sequences was 2 906 bp altogether. Homology analysis showed that buffalo NOBOX gene shared 99%,96%,91%,and 91% of similarity with that of the Mediterranean Sea buffalo,cattle,sheep and goats,respectively. The predictions for the promoter and transcription factor- binding site of 2 000 bp of the 5'flanking sequence of the NOBOX gene showed that there was a TATA box at the- 868 /- 853 bp site on the upstream of the translation initiation site,and there were GATAs,Foxo1,Foxo3,Nobox,Stat1,Stat3,Stat4,Stat5 a,Stat5b,Stat6,YY1,and other binding sites of trans- acting elements in the promoter region,thereinto,GATAs family genes existed many binding sites in the promoter region of NOBOX gene,and there were several combinations of GATA genes in the same site of NOBOX promoter. The promoter of buffalo NOBOX gene could start enhanced green fluorescent protein( EGFP) expression in HEK- 293 T cells,while the expression is very weakly. It could also start EGFP expression in CHO cells,and the expression intensity was similar with that of CMV starting the expression of EGFP in CHO cells. The results indicate that the promoter of buffalo NOBOX gene is a strong promoter.