Study On Transcriptional Regulation Of Hu Sheep BMP7 Promoter

In previous study,BMP7 was considered to be a candidate gene by correlation analysis of gene chip and the histology characteristics of hair follicle between different patterns in Hu sheep.As we all know,BMP7 is one of the largest secretory signal conductive molecule which is in TGF-? superfamily.BMP 7 is not only involved in bone formation,but also found to have a great deal with the size and spatial distribution of feather germ,and play an important role in the growth and development of hair follicle,however,the regulation mechanism is not clear.For the purpose of preliminary study on regulation mechanism of BMP7 promoter,we firstly analyzed the expression of BMP7 in different tissues of Hu sheep.Then,proximal promoter of BMP7 was cloned for bioinformatics analysis.Next,a series of missing vectors has been constructed for double fluorescein activity detection according to bioinformatics analysis results and the promoter sequence of the proximal promoter.Finally,we tested transcription factor binding site mutations and transcription factor over-expression were as the foundation for further research to reveal the transcriptional regulation principle of BMP7 promoter region.In this study,the transcriptional regulation mechanism of BMP7 was studied from the following aspects:(1)The expression of BMP 7 in the tissues of Hu sheep has been analyzed by RT-qPCR,and it was found that BMP 7 was high expressive in hair follicle tissue.(2)We has got the control sequence from 2000 bp upstream to 500 bp downstream of the BMP7 proximal promoter,and obtained 1757 bp sequence by PCR cloning.The upstream transcriptional regulatory region of BMP7 gene's proximal promoter was predicted by bioinformatics technique.It was found that there were-1216bp—-1166bp and-632bp—-582bp transcription initiation sites in the upstream transcriptional regulatory region of BMP 7 gene's proximal promoter.Based on the analysis of CpG island's distribution,we found that there were many CpG islands at-549bp—+295bp.With the prediction of transcription factor binding site,the transcription factor binding sites such as SP1,EGR1,NRF1 and TFAP2B have been found.(3)According to the sequence of BMP7 gene's proximal promoter,a series of deletion fragments were designed,with the double luciferase reporter gene vector constructed.293T cells were transfected through double-luciferase to detect the activity of different deletion fragments and take significant comparison for the highest activity of sequence fragments.It was found that there was a high degree of activity between-758bp and-545bp,and the transcription factor SP1 and EGR1 may have binding sites to regulate the BP7 promoter with bioinformatics prediction.(4)In order to further identify the transcription factor binding sites in the transcriptional regulatory region of the proximal promoter of the BMP7,EGR1 and SP1 were screened according to the bioinformatics results.with the site-directed mutations in their transcription factor binding site.At the same time,At the same time,constructing the BMP7 gene's transcription factor EGRI for over-expression Carrier verification.The results showed that the transcriptional activity of the BMP7 was significantly decreased in the transcriptional regulatory region of the BMP7 after mutation,and significantly enhanced by the over expression of EGR1 transcription factor,which preliminary revealed that EGR1 and SP1 play an important role in BMP7 regulation.