Cloning, Expression And Polymorphism Analysis Of BMP4 Gene In Hu Sheep

Hu sheep of our country was world-famous for its hyper-prolificacy performance, but the heredity mechanism was not explicit. Bone morphogenetic protein 4 (BMP4) was a member of bone morphogenetic protein subfamily, which belongs to TGF-βsuperfamily. Researches indicated that BMP4 plays a crucial role on reproductive function in Mammals, such as promoting follicular development and oocyte maturation, increasing ovulation rate.In this study, BMP4 DNA sequence of Hu sheep was cloned for the first time, and gene structure, sequence and phylogenetic analysis were carried on using bioinformatic methods.We also analyzed tissue-specific expression using RT-PCR; the relationship between gene expression level in ovaries, ovulation rate and litter size were analyzed using real-time PCR; gene polymorphism was detected using PCR-SSCP, different genotypes of BMP4 and litter size of Hu sheep was analyzed too. Here, we take BMP4 as a candidate gene, to find its relationship with reproduction performance of Hu sheep, hope to provide the reference or even revealing the molecular mechanism of hyper-prolificacy in Hu sheep. The results as follows:1. Gene clone and sequence analysis of BMP4 in Hu sheepBMP4 DNA sequence was cloned 4694 bp in Hu sheep, which in composed of 4 exons and 3 introns, including 2 non-coding exons. The coding sequence was 1230 bp, which composed by exon 3 and exon 4, encoding 409 amino acids. There were 4α-helix and 8β-sheet in Hu sheep BMP4 protein which predicted by analysis of space structure. BMP4 protein contains two conserved structure domains:TGFβ-propeptide and TGFβ. The location and sequence of TGFP-propeptide domain were highly conserved in mammals, and TGFβdomain was entirely conservative. The phylogenetic tree made by BMP4 amino acid sequence using neighbor-joining was consistented with the classical taxonomy of species. In viewing of the high gene homologous in mammals, it suggested that BMP4 plays an important role on follicular development in Hu sheep as it does in other mammal species.2. BMP4 gene expression level in ovaries with reproduction performance in Hu sheepThe tissue expression of BMP4 was detected using RT-PCR. The results showed that BMP4 mRNA existed in hypothalamus, pituitary, oviduct, uterus, heart, liver, spleen, lung, kidney and muscle of Hu sheep. The expression level of BMP4 mRNA in ovaries was detected using real-time PCR, and the relationship with ovulation rate and litter size of Hu sheep were analyzed. The results showed that the expression level of BMP4 mRNA and ovulation rate was significantly positively correlated (P<0.01), the correlation coefficient was 0.8663, gene expression level (Y) and ovulation rate (X) linear regression equation:Y =-0.3213+0.2362X (R2=0.7505); the expression level of BMP4 mRNA and the first litter size was significantly positively correlated (P<0.01), correlation coefficient was 0.8861, with the linear regression equation:Y=-0.3701+0.4734X (R2=0.7851); the expression level of BMP4 mRNA and the sceond litter size was significantly positively correlated (P<0.05), correlation coefficient was 0.7567, with the linear regression equation: Y=-0.0291+0.1845X (R2=0.5726); the expression level of BMP4 mRNA and the average litter size was significantly positively correlated (P<0.01), correlation coefficient was 0.8744, with the linear regression equation:Y=-0.1753+0.2862X(R2=0.7645); Our research indicated that close relation existed between BMP4 gene and the reproductive performance, so BMP4 may be a candidate gene for high fecundity in Hu sheep. 3. Gene polymorphism of BMP4 and its effect on little size in Hu sheep220 Hu sheep were detected for BMP4 gene polymorphism using PCR-SSCP. The products were amplified by primer T2 detected two genotypes:AA and AB. Compared with genotype AA, a A→C mutation occurs at 287bp of BMP4 coding sequence for genotype AB, leading to the 100th amino acid change from isoleucine (Ile) to leucine (Leu). In the population of Hu sheep, AA genotype was the dominant type with frequency of 0.7758; A was the dominant allele gene with frequency of 0.8794. We found the trends that different genotypes showed AA>AB on little size:the third and multiparity average little size with genotype AA was significant higher 0.86 and 0.57 more lambs respectively than that of AB (P<0.05). Our research suggested that the A298C sites of BMP4 may have effort on litter size, BMP4 can be used as an assistant selection marker for breeding in Hu sheep.