The Cloning And Functional Analysis Of BTG2Promoter Region Of Mongolian Cattle And Buffalo

BTG2is a recently identified anti-proliferative gene which belongs to the BTG/TOB gene family. It is a transient primary-response gene and encodes a NGF-inducible secreted protein. Species and individual expression differences of BTG2exist in pig and cattle. To explore the difference between genes expression as a result of various molecular mechanism in buffalo and Mongolian Cattle, our study focused on the promoter region of the BTG2in these two mammals. Our results showed:Using the DNA template from the buffalo and Mongolian Cattle, we cloned the promoter region of the BTG2, Buffalo (1478bp) and Mongolian Cattle (1708bp). Comparison of these two sequences showed a lack of269base pairs in the buffalo promoter DNA sequence. Furthermore, bioinformatic analysis revealed multiple transcription factor binding sites from the deletion fragment, and this laid the foundation for the future studies on the analysis of gene polymorphism and function.Promoter sequences were recombined into pEGFP-N1which is a mammalian expression vector, and the transient expression vector pBTG2(p)-EGFP was confirmed by sequencing analysis. NGF induced HeLa cells with pBTG2(p)-EGFP, GFP reporter gene expression was detected in all HeLa cells, and the transcriptional activity of Mongolian cattle BTG2promoter is much higher than buffalo. The root cause of this phenomenon is perhaps the lack of269bp fragment in the buffalo BTG2promoter.To study whether or not the lack of269bp fragment in the buffalo BTG2promoter has an impact on transcription, the expression of the BTG2in buffalo and Mongolian cattle muscle tissue were amplified by qRT-PCR. Comparison of these two results showed Buffalo BTG2expression was significantly reduced. This indicates that the lack of269bp DNA sequence is likely to be directly related to the BTG2promoter activity.It was also found that Mongolian cattle nucleoprotein interacts with the deletion fragment in BTG2promoter region. EMSA results showed DNA content in the same conditions as the nucleoprotein concentration is increased, although the DNA1and nucleoprotein binding rate is on the rise. This study speculated that the DNA fragment may contain some specific cis-elements that associated with meat quality or cold tolerance.The studies suggest that BTG2is likely to be a candidate gene in assessing beef quality, cold and drought tolerance, which laid the foundation for subsequent research of BTG2.