湖羊lncRNA-269的鉴定及转录调控分析OACSTPCD

[Objectives]The paper aimed to study the sequence characteristics,expression patterns and transcriptional-regulation of lncRNA-269 of Hu sheep in order to provide theoretical basis for further research on its function.[Methods]Taking Hu sheep as the object,the full length of its sequence was obtained by 5′/3′RACE combined with PCR.RT-qPCR was used to identify its expression pattern in Hu sheep tissues;its promoter sequence was amplified using PCR method;the characteristics of its promoter region was identified,and its transcriptional regulation was analyzed.[Results]The total length of lncRNA-269 in Hu sheep was 3 146 bp,which was widely expressed in various tissues of Hu sheep,and significantly expressed in healthy follicles.Its expression level was positively correlated with NR5A1 mRNA and serum E2 level.The luciferase activity analysis showed that there was a transcriptional activation motif between-359 and-17 nt in lncRNA-269 promoter region,and a transcriptional inhibition motif between-1 485 and-942 nt.JASPAR software predicted that some transcription factor binding sites such as FOXO3 and PDX1 existed in transcriptional inhibition region,and SMAD4 and YY1 existed in transcriptional activation region.The overexpression of SMAD4 significantly increased the luciferase activity of lncRNA-269,and chromatin immunoprecipitation(ChIP)and PCR(ChIP-PCR)assay further confirmed the specificity of SMAD4 binding to lncRNA-269 promoter region.[Conclusions]lncRNA-269 was highly expressed in the healthy follicles of Hu sheep.There was a transcriptional activation region and a transcriptional inhibition region in its promoter region.The transcription factor SMAD4 could significantly up-regulate the promoter activity of lncRNA-269 by binding with the lncRNA-269 promoter region.