Cloning And Expression Study Of Bovine Bone Morphogenetic Protein-15 Gene

Bone morphogenetic protein-15(BMP-15), which belongs to transforming growth factorβ(TGF-β)super family, is shown to be essential for regulating growth and development of ovarian follicles and female reproduction. In the present study, part of cDNA fragment of bovine BMP-15 gene was cloned by RT-PCR based on sequence information of other mammalian BMP-15 genes. Then expression of BMP-15 in bovine ovaries at mRNA level was examined by in situ hybridization (ISH) technique. Methords: Firstly, searching for BMP-15 gene sequencese of sheep and mouse in Genbank, and homology of BMP-15 gene sequencese was analysed using DNAStar software. According to the homology of the sequencese, specific primers were designed. Total RNA was extracted from bovine ovaries and part of BMP-15 cDNA was amplified by RT-PCR, then PCR product was recombined into pGM-T vector. After restriction endonuclease digestion and PCR analysis, the recombinant plasmids were sent to sequencing. Secondly, recombinant plasmid DNA was amplified using T7 and SP6 primers by PCR, and the PCR product was used as tamplate to produce DIG labelled cRNA probe. Expression of BMP-15 mRNA in bovine ovaries was examined with DIG-cRNA probe by ISH technique. Results: a 423 bp cDNA sequence was obtained by RT-PCR from bovine ovaries. On the bases of comparing the sequence to goat, sheep, pig, dog, human, mouse and rat, the fiding homology were 98%, 97%, 90%, 89%, 85%, 78% and 77% respectively, which confirmed the cDNA as bovine BMP-15 cDNA. The results of ISH indicated that BMP-15 mRNA was expressed in bovine ovaries and the location of BMP-15 mRNA differed due to different follicle dedveloping stage: in primary and early secondary follicles, signals were located mainly in granulosa cells, and in late secondary follicles, signals were located solely in zona pellucida.