Cloning, Sequencing And Polymorphisic Studying Of Pig Bone Morphogenetic Protein-15 Coding Sequence

Bone morphogenetic protein-15 (BMP 15) is a growth factor secreted by oocytes in growthing ovarian follicleso BMP15 gene is expressed only in the oocytes.BMP15 is essential for growth and differentiation of early ovarian follicles and ovulating. Otsuka et al. proposed that granulosa cells (GCs) were the first target cells for BMP15 and the biological significance of oocyte-specific BMP15 might be to promote follicle growth, while preventing premature luteinization. In that, we guessed that BMP15 is essential for female fertility and that natural mutations in an ovary-derived factor can cause both increased ovulation rate and infertility phenotypes in a dosage-sensitive manner.This study was established on comparing genonlics.The project of this study was to expect to attain the coding sequence of pig BMP15 gene and to establish a suitable method of polymorphisim.The sample materials were Large White and MeiShan pig.According to the BMP 15 sequeces of human ,rat and pig , four pairs primers were designed and synthesized by comparing genomics.The genome DNA was amplified using the primers.Then.the product was cloned and sequenced.After that,the DNA sequences were sequenced using BLASTN soft.(l)The complete coding sequences including exon land exon 2 of pig BMP15 were obtained while they were submitted to GenBank (Accession Number, Large White: AH012831,Mei Shan: AH012830).(2)Comparing the coding sequences of Large White and Meishan pig BMP15 by PairwiseBLAST,a new SNP was found in the area of exon 2.The SNP was at the point of 390th nucleotide of the coding sequences. At this point,the nucleotide of Large White pig was T while Mei Shan pig of that was A.The SNP resulted in the change of the digesting site of restriction endonuclease Spel.(3)Basing on Spel,BMP15 PCR-RFLP was performed among Duroc,Largewhite and Meishan pig.BMP15 gene had three genotypes:BMP15AA,BMP15AB and BMP15BB.(4)According to PCR-RFLP, polymorphism was studied between Duroc, LargewhiteI, LargewhiteII, Landrace, DIV1 , DIV2, Erhualian and Mei Shan .the genotype frequencies and allele frequencies of BMP15 were significantly different. Allele "A" frequency in Duroc, Largewhitel, LargewhiteII,Landrace ,DIV1and DIV2 pig was zero while allele "B" frequency was 100 percent.(5)Using SAS 8.0, significant association between BMP15 and reproduction performance was found. The total number born (TNB) and number born alive (NBA)of first parity and later parities were significantly different between lines(p<0.01).Cloning,sequencing and sequencing analysis of BMP15 gene gave us some ideas of the real function of BMP15, while it is important to pig breed and improving TNB.