Cloning And Polymorphism Analysis Of GDF9 Gene And BMP15 Gene In Hu Sheep And Bashibai Sheep

GDF9 and BMP 15 gene are two major genes of sheep high prolificacy trait,both of them are specifically expressed in ovarian oocytes of follicles,and play an important role in regulating the growth of ovarian follicles and proliferation of cumulus cells and ovulation.At present,a number of SNPs were found in GDF9 gene and BMP 15 gene coding regions that were significantly associated with the high prolificacy of overseas sheep breeds,but these SNPs were not significantly associated with Hu sheep.In order to further define the relationship of GDF9 gene and BMP 15 gene with the high prolificacy in Hu Sheep,Hu sheep and Bashibai sheep were the subject of this study,we obtained whole genome of GDF9 gene and coding regions and 5’UTR of BMP15 gene,then analysis the sequence features of them;Screening SNP of GDF9 gene and BMP 15 gene in the two sheep breeds;Analysis of the relationship between SNP with Hu sheep litter size;The effect of SNP on promoter activity was investigate using dul-luciferase reporter assay system.Our results provide theoretical basis for exploring the potential relationship between GDF9 gene and BMP 15 gene with reproductive performance of sheep and screening moleesular markers of high prolificacy in Hu Sheep.Results as below:(1)Genomic sequence features of GDF9 in Hu sheep and Bashibai sheepGDF9 genomic sequence was cloned 5150 bp in Hu sheep and Bashibai sheep,which in composed of s’UTR、2 exons、1:intron and 3’UTR.There are three potential promoter regions were predicted in GDF9 gene 5’UTR,which are located-1129nt--1079nt,-633nt-583nt and-270nt--220nt.The Bioinformatics found that there are transcription factors binding sites such as GATA4、SOX8、YY1、HSFI、API1、OCT1、SOX1 and PTF1 in GDF9 gene 5’UTR.Besides,GATA4、YY1 and HSF1 have been shown to correlate with follicular development.The coding sequence of GDF9 gene was all 1362 bp in Hu sheep and Bashibai sheep,which encoding 453 amino acids,has high sequence identity with other mammals.It contains typical structural domains such as TGFp,and M1-S32 amino acids was the potential signal peptide sequence.There were 2 α-helix and 6 β-sheet in Hu sheep and Bashibai sheep GDF9 protein which predicted by analysis of space structure.The Bioinformatics found that there are five miRNA binding sites,miRNAs such as niR-515-5p、let-7e-3p and let-7a-2-3p,and these three miRNAs all correlate with follicular development.(2)Relationship between GDF9 gene SNPs polymorphism with litter size in Hu sheepFive SNPs of GDF9 gene were screened using DNA pool technology and were detected using sequencing method,including one SNP(1599A>G,did not change amino acid)in coding sequence,three SNPs in 5’UTR(-534A>G、-407T>G and-332C>T,-534A>G change transcription factor binding site of OCT1)and one SNP(2547A>G,did not change miRNA binding site)in 3’UTR.The three SNPs of GDF9 gene promoter was Complete linkage in Hu sheep and Bashibai sheep,and called the wild haplotype A-T-C as A,mutant haplotype G-G-T as B,There were three genotypes(AA,AB,BB)of GDF9 gene in Hu sheep and Bashibai,AA genotype was the dominant type with frequency of 0.6292 and 0.6420 in Hu sheep and Bashibai sheep,respectively;A was the dominant allele gene with frequency of 0.7921 and 0.7840,respectively.Association analysis showed that,the litter size of AA.AB genotype was significantly higher than the BB genotype.The luciferase reporter assay indicated that the promoter activity of genotype AA was no significantly different with genotype BB.But when they was co-transfected with the overexpression of the transcription factor OCT1,the promoter activity of genotype BB was decreased more significantly than genotype AA.The results suggest that the SNP(-534A>G)may effect the promoter activity of GDF9 gene through the transcription factor OCT1.Annexin v FITC/PI flow cytometry results showed a significantly increase of GC apoptosis after Overexpression of OCT1 treatment,suggest that the transcription factor OCT1 can promote the apoptosis of granulosa cell.(3)Genomic sequence features of BMP15 in Hu sheep and Bashibai sheepWe obtained the coding region of BMP 15 gene by cloning and sequencing technology in Hu sheep and Bashibai sheep,respectively,and the full length is 1182bp(the homology of both was 99.92%),encoding 393 amino acids(the homology of both was 100%),and has high sequence identity with other mammals.It contains typical structural domains such as TGFβ,and M1-M24 amino acids was the potential signal peptide sequence.There were 2α-helix and 6 β-sheet in Hu sheep and Bashibai sheep BMP 15 protein which predicted by analysis of space structure.We obtained the 5’UTR of BMP 15 gene by cloning and sequencing technology in Hu sheep and Bashibai sheep,respectively,and the length is all 1806bp.Here is one potential promoter regions were predicted in BMP 15 gene 5’UTR,which is located-1617 nt--1568 nt.The Bioinformatics found that there are transcription factors binding sites suchas HSF、CdxA、OCT-1、SRY、SOX-5、GATA-1、AP-1、NIT-2 and GATA-2 in BMP15 gene 5’UTR,and HSF、OCT-1 and SP1 have been shown to correlate with follicular development.(4)c.-1760C>A variant of BMP 15 gene and its association with the promoter activity in Hu sheep and Bashibai sheepSix SNPs of BMP 15 gene were screened using DNA pool technology and were detected using sequencing method,including one SNP(6355G>A,did not change amino acid)in coding sequence,five SNPs in 5’UTR(-1760C>A、-1548G>A、-1146C>T、-1145G>A and-609G>A).About c.-1760C>A,there was one genotype(CC)in Hu sheep and three genotypes(CC,CA,AA)in Bashibai sheep,genotype frequency was 0.6154、0.3654 and 0.0192,respectively.The luciferase reporter assay indicated that the promoter activity of genotype CC was obviously high than genotype AA,but did not up to significant level(P=0.069).The result suggest that the SNP(c.-1760C>A)may be involved in the effect on the promoter activity of BMP 15 gene.