Cloning And Characterization Analysis The5’ Regulation Fragment Of Buffalo Bone Morphogenetic Protein-15and Growth Differentiation Factor-9Gene

To investigate the expression pattern of BMP-15and GDF-9gene in water buffalo during follicular genesis, the regulation region of BMP-15and GDF-9was cloned and the function of the DNA fragments were verified in cell level. According to the genome sequence of bovine, primers of BMP-15and GDF-9were designed. Using the local buffalo genomic DNA as templates, different length of BMP-15and GDF-9regulation sequences were amplified by PCR. After correctly sequencing analyzed, these fragments were inserted into pEGFP-1vector, and EGFP reporter vectors were successfully generated. Then. these vectors were transfected into cells derived from different tissues, the ability of regulation fragments and tissue specific expression profiles were detected, results as following:1. The5’ regulation region of buffalo BMP-15gene was cloned. The length of the cloned fragment was4.6kb, including a2.8kb sequence upstream of transcription start site and a1.8kb sequence down stream. The sequence upstream of transcription start site of the buffalo was98%similar to that of the cattle. In order to analysis the transcription ability of different section in the promoter, five different fragments were amplified by subsection clone, including B4.6, BTL, B2.9, B1.6and B1.3, the five fragment were reconstructed as pBMP-15-EGFP-1respectively. After transfected into different cell types, results showed that only BTL (-2788～+219) could express EGFP in LBT2cell water buffalo granular cell, CHO cell and SKOV3cell, but not in buffalo fetal fibroblast cell and pig fetal fibroblast cell.2. The5; regulation region of buffalo GDF-9gene was cloned. The length of the cloned fragment was3.7kb, including a2.3kb sequence upstream of transcription start site and a1.4kb sequence down stream. The sequence upstream of transcription start sit of the buffalo was98%similar to that of the cattle. And, In order to analysis the transcription ability of different section in the promoter, five different fragments were obtained by subsection clone, including G3.7, GTL, G2.7, G1.1and G1.21, the five fragment were reconstructed as pGDF-9-EGFP-1respectively. After transfected into different cell types, results showed that only GTL (-2230～+89) possess the obviously transcription ability, EGFP were successfully expressed in LBT2cell, water buffalo granular cell, CHO cell and SKOV3cell, but not in buffalo fetal fibroblast cell and pig fetal fibroblast cell.In conclusion, water buffalo BMP-15and GDF-9gene promoter activity region are located in the BTL (-2788～+219) and GTL (-2230～+89),and these regulation fragments could control the EGFP expressing in LBT2cell, water buffalo granutar cell, CHO cell and SKOV3cell, but not in buffalo fetal fibroblast cell and pig fetal fibroblast cell, suggest that the two regulatory sequences should be the tissue-specific promoter.