Cloning And Expression Of Bone Morphogenetic Protein-4 In Huanghuai Goat

The experiment was carried out to study the prolific trait, the genetic diversity, the local variety resources and the species diversity, and to provide evidence for variety breeding and industrialized development of HuangHuai Goat.The encoding sequence,Intron2,3 and partial promoter of BMP-4 cloned by gene recombination and optimizedly express the mature peptide in E.coliBL21(DE3)plysS. And the Tissue-spe -cific expression of BMP-4 investigated by RT-PCR.The following conclusion were obtained:1 The cDNA sequence that including the integrity open reading frame(1230bp) was cloned from kidney of HuangHuai Goat by RT-PCR. The sequence has been submitt -ed to GeneBank and the accession number is EF632080.To analysis the homology of the sequences.Corresponding sequence alignment of goat with sheep,pig,human,mouse and cattle demonstrated that the homologies of the nucleotides were 99.43%, 96.10%,94.96%,91.79%,99.19% respectively. This indicates that BMP-4 is highly con -servative in the species. Comparing with human and mouse,goat has more 3bp,and the homology of goat,sheep and cattle are over 99%,this suggest that the genetic relationship of goat,sheep and cattle are closer than other species.Goat BMP-4 codes 409 amino acid(aa) that including signal peptide(19aa), propeptide(274aa) and mature peptide(116aa), theoretical MW is 46.63 kDa.The mature peptide has the typical characteristics of TGF-β:seven cysteine residues. The aa homology is 99.51%,98.53%,97.80%,98.04%,99.51% respectively,this show that the genetic relationship of goat,sheep and cow are closer than other species too.But which is the closer relationship with goat between sheep and cattle need the further research.2 Primer design with extron-extron,we get the 1053bp and 962bp sequence of intron2 and intron3 of BMP-4(the NCBI accession number is EU104684).The sequences has the general feature of intron-"GT-AG",which indicates that the obtained introns are complete.To analysis the intron of pig,human,mouse and cattle,the length of intron2 and intron3 are 1028bp,1046bp,1061bp,1062bp and 949bp,964 bp,1002 bp,961bp respectively. Corresponding sequence alignment of goat with pig,human, mouse and cattle demonstrated that the homologies were 83.80%,76.03%,66.94%, 96.71% and 87.15%,80.24%,67.20%,98.13% respectively.The length of the goat partial promoter is 676bp(the NCBI accession number is EU679369). 3 The cDNA encoding the mature peptide was amplified and cloned into pET32avector, the E.coliBL21(DE3)plysS was transformed and expressed with recombinant plasmid.The MW of recommend protein is 31 kDa and the mature peptide is 13.1 kDa. Optimization of Conditions for expression according to the expressing quantity,and the optimized conditions is 0.5mMIPTG, 22℃,induced 8h. The subcellular localization for recommend protein reference to the《Molecular Cloning》,which shows that the main form of recommend protein is inclusion body, cytoplasm and periplasm have a little, but culture medium has no.4 To test the tissue-specific expression of BMP-4 mRNA in the seven tissue.The result shows that BMP-4 expressed in all tissue,the high expressed tissue is kidney,ovary and spleen, the heart,lung,liver take second place, the pancreas high express lowly.In conclusion that the expresion of BMP-4 mRNA has tissue specificity.