Cloning,Polymorphism And Expression Analysis Of BMP4 Gene In Porcine

Bone morphogenetic protein 4(BMP4)is an important member of the transforming growth factor-?(TGF-?)superfamily.It is essential for the survival of primordial follicles and oocytes.And it is closely related to the reproductive performance of mammals.At present,there are few studies on the BMP4 gene of porcine,and the research on the polymorphism has not been reported.In this study,we use PCR amplification and cloning and sequencing techniques to obtain the Suhuai pigs and Meishan pigs BMP4 genomic sequence,and using bioinformatics methods to analyze its genetic characteristics analysis.Using the pool DNA sequencing to screen Suhuai pigs and Meishan pigs BMP4 gene SNP locus,and AS-PCR in Suhuai pigs and Meishan pigs population BMP4 gene SNP locus.Using the RT-PCR technique to analyze the tissue expression characteristics of porcine BMP4 gene,and cloned into eukaryotic expression vector pcDNA3.1 to construct the BMP4 gene over expression vector.The results provide reference for revealing the characteristics and biological function of BMP4 gene in porcine.The results are as follows:1.Cloning and sequence analysis of BMP4 in porcineBy PCR amplification and cloning and sequencing techniques to obtain Suhuai pigs and Meishan pigs BMP4 genome sequence 7147bp,consistency between the two nucleotide sequences is 98.63%.Electronic chromosomal localization of porcine BMP4 gene located on porcine chromosome 1,between 203773 kb?203781 kb.Genomic structure analysis of porcine BMP4 gene consists 4 exons and 3 introns.We discovered a potential promoter region in Suhuai pigs and Meishan pigs BMP4 gene 5'terminal-1051?-801nt,including HEN1,STAT1 and AP4 transcription factor binding site,but no typical TATA box and CAAT box are found in the eukaryotic promoter.Suhuai pigs and Meishan pigs BMP4 gene 3'-UTR sequence length 267bp.Its homology is 100%,containing AC repeats,tail signal,PolyA structure and conservative ARE ect.Suhuai pigs and Meishan pigs BMP4 gene coding region only contains two exons(exons 3 and exons 4).Its full-length sequence is 1230 bp,encoding 409 amino acid residues,both the nucleotide and amino acid sequence identity 100%,and the consistency with other mammalian species,respectively 91%and 97%.Like other mammals,Suhuai pigs and Meishan pigs BMP4 protein also contains a typical pre-TGF-? peptide and TGF-?-like domain.The results show that Suhuai pigs and Meishan pigs BMP4 gene and other mammals in genomic structure,sequence and protein structure with high conservation.2?3'UTR polymorphism analysis of BMP4 gene in porcineUsing the pool DNA sequencing technology,we find a SNP in Suhuai pigs and Meishan pigs BMP4 gene 3'UTR 20nt,G mutation to C,and named it as*20G>C.Using established AS-PCR method to detect the Suhuai pigs and Meishan pigs population BMP4 gene*20G>C of polymorphic locus,as a result in Suhuai pigs and Meishan pigs populations are detected in GGQ GC and CC genotypes.In Suhuai pigs population,GG genotype and G allele are the advantage genotype and the dominant allele,respectively.The frequency are 0.815 and 0.887.On the contrary,in high fecundity breeds Meishan pigs,CC genotype and C allele are the advantage genotype and the dominant allele,respectively.The frequency are 0.667and 0.770.Bioinformatics methods predict that the BMP4 mutation of porcine*20G>C gene resulted a change of miR-7135 binding site.Therefore,in this article we constructed two kinds of luciferase containing the polymorphic site of the expression vector(pmirGLO-G and pmirGLO-C),and miR-7135 mimics co-transfected with 293 cells,luciferase activity analysis showed no significant difference in different luciferase expression vector activity,BMP4 gene*20G>C mutation may not affect the combination of miR-7135.The results showed that the*20G>C polymorphism of BMP4 gene may be related to the reproductive performance of pigs.3?The expression profile and eukaryotic expression of BMP4 gene in Suhuai pigsThe expression characteristics of Suhuai pigs BMP4 gene are analyzed by using RT-PCR method.The results showed that the BMP4 gene is expressed in tissues of Suhuai pigs' heart,muscle,ovary,liver,kidney and spleen.Besides,we find that BMP4 gene is highly expressed in the heart,muscle,ovary and kidney tissues while is less expressed in liver and spleen tissues.By cloning the coding region of pig's BMP4 gene into pcDNA3.1 vector,BMP4 gene eukaryotic expression vector can be constructed.And after comparing enzyme digestion identification and plasmid sequencing,the result shows that the anticipated sections connected to the vector sequence is completely correct.From which,we know that pig's BMP4 gene eukaryotic expression vector was successfully constructed Therefore,we call pig's BMP4 gene eukaryotic expression vector "pcDNA3.1-BMP4".The recombinant plasmid pcDNA3.1-BMP4 transfection in vitro cultured porcine granulosa cells,Western blot analysis showed significant BMP4 protein bands at the 23KD granule cells transfected with recombinant plasmid pcDNA3.1-BMP4 group,BMP4 protein expression levels significantly the control group,indicating that pcDNA3.1-BMP4 recombinant plasmid capable of high expression in porcine ovarian granulosa cells,laid a foundation for further study its function.