Identification And Regulation Of The FSHR And TGF-β1 Gene Core Promoter Region In Hu Sheep

Hu sheep is one of the unique high fecundity varieties in China, at the same time it’s also one of the 138 national key protected local varieties. It comes from Taihu, with the all year rut,high reproduction early rapid growth, early maturity, resistance to heat, appropriate feeding tender and delicious meat performances. Therefore, Reveal the molecular mechanism of Hu Sheep traits formed, and screening the candidate genes and functional SNP are very important for the conversation and innovation of Hu sheep unique multiple births trait. Hu sheep is the subject of this study. We obtained coding regions of FSHR and TGF-β1 gene in Hu sheep by PCR amplification and cloning techniques, and then analysis the sequence features with bioinformatics software; we cloned the 5’regulating region and constructed deletion expression vectors to screen its core promoter by luciferase reporter gene systemer, and then identified the SNP in the core promoter region by sequencing between prolificacy group and single group, and analysised the expression and regulation of the promoter region. in order to provide a basis to improve the high fecundity. Results as below:(1)By cloning and sequencing technology on the FSHR, we obtained the coding region of Hu sheep FSHR gene, and the full length is 2088bp. The protein FSHR consists 696 amino acid residues.We found that amino acid sequence homology with other mammals is 75%-97%.It contains typical structural domains sunch as LRR. It showed that FSHR gene of Hu sheep is relatively conservative in envolution with other mammals. We presumed that FSHR gene of Hu sheep plays an important role in follicular development as other mammals.(2)We obtained the 5’flanking sequence by cloning and sequencing technologies and the length is 1920bp. We constructed three deletion vectors (pFSHR-497, pFSHR-735, pFSHR-1459) and then transiently transfected in COV434 and COS-7 cell. The results showed that the luciferase activity of the pFSHR-735 is significantly higher than other vectors. So the core promoter region of FSHR gene locate at-505nt--743nt. The Bioinformatics found that there are transcription factor binding sites such as YY1, AP-1, USF, Spl and HSF. By direct sequence the -575A>G in the core promoter region was detected.(3) By sequencing between prolificacy group and single group we found a SNP in the core promoter region -575nt,-575A>G mutation. To futhur study the effect of the -575 A>G in the core promoter region on the activity of the promoter, the vectors of wild type (AA) and mutated type (GG) were constructed (pFSHR-AA and pFSHR-GG) and then transiently transfected in COV434 and COS-7 cells. It showed that compared with the wild type the activity of the core promoter the mutated type significantly reduced (P<0.05). The Bioinformatics found that there is an YY1 transcription factor binding site in the core core promoter region of Hu sheep FSHR gene. In order to verify the regulation of transcription factor YY1 on the FSHR gene transcription, we co-transfected the over-expression vector pUC57-YY1 and the pFSHR-AA into the COV434 cell. The luciferase activity found that after over-express the transcription factor YY1, the activity of the core promoter is significantly highly(P<0.01).-575A>G mutation in the core promoter region made the mutated type add a new CpG. To analysis the effect of the DNA methylation on the activity of the mutated type promoter, we modified GG-type expression vector in vitro methylation by M. SSSI methyl transferase and made the CpG site methylation. Then we transiently transfected into the COV434 cells. We found that the expression of the methylation vectors activity reduced significantly (P<0.01). The results showed that the activity of the Hu sheep core promoter region is regulated by the mutation, transcription factor YY1 and DNA methylation.(4) By cloning and sequencing technology on the TGF-β1, we cloned the coding region of Hu sheep TGF-β1 gene. The result showed that the full length of the coding region is 1173bp. The protein TGF-β1 consist 390 amino acid residues. The amino acid sequence homology with other mammals is 89%-99%. It contains typical structural domains sunch as TGF-P proeptide(Ala28-Met261), TGF-β like (Glu290-Ser390) and so on. It showed that TGF-β1 gene of Hu sheep is relatively conservative in envolution with other mammals. We presumed that TGF-β1 gene of Hu sheep plays an important role in follicular development and ovulation as other mammals.(5) We obtained the 5’flanking sequence by cloning and sequencing technologies and the length is 1571bp. We constructed four deletion vectors (pTGF-β1-188, pTGF-β1-341, pTGF-β1-540, pTGF-β1-1571) and then transiently transfected in COV434 cell. The results showed that the luciferase activity of the pTGF-β1-1-540 is significantly higher than other vectors. So the core promoter region of TGF-β1 gene locate at -638nt～-439nt. The Bioinformatics found that there are transcription factor binding sites such as HSF, Sp1, AP-2, ADR1 and HSF2. It’s a pity that there is no SNP in the TGF-β1 core promoter region by sequencing.