Cloning, Radiation Hybrid Mapping, Promoter Regulation And Genetics Effect Analysis Of Porcine ADAMTS1 Gene

A disintegrin-like and metalloprotease(reprolysin type) with thrombospondin type 1 motif(ADAMTS) is a novel family of extracellular proteases found in both mammals and invertebrates.Later research showed that ADAMTS1 plays an important role in follicles via progesterone receptor(PR)-dependent pathways.Female fertility is impaired in ADAMTS1 knocked out mice,and this is accompanied by obvious abnormalities of the uterus and ovaries.In addition,there is evidence that progesterone- and PR-dependent functions in cumulus cells are essential for cumulus-oocyte complexes(COC) expansion in pig,possibly through the action of ADAMTS1.All these data suggest that ADAMTS1 plays a critical role in follicular rupture and represents a major advance in the proteolytic events that control ovulation.In this research,the ADAMTS1 gene were cloned and identified and the promoter regulation and genetics effect were analyzed.The main results are as follows:1.The sequences of cDNAs of human and mouse ADAMTS1 genes were used to search against the porcine EST databases.Six porcine ESTs sharing＞80%identity to the corresponding human and mouse cDNAs were obtained and assembled into a contig. Based on the contig sequence,a set of primers was designed to amplify the DNA sequence of porcine ADAMTS1 gene.These primers yielded twelve overlapping PCR products that produced a consensus sequence of 9026-bp DNA sequence containing the full coding region,all 8 introns and part of 5'and 3' untranslated region of the porcine ADAMTS1 gene was obtained(Genbank accession number DQ177331).2.Using CLUSTAL W and some related software;we analyzed the gene structure, protein structure and conserved motifs.The hydropathy plot and the TMAP prediction derived from the World-Wide Web service showed that porcine ADAMTS1 protein includes no transmembrane region and there are many cysteine residues and four putative N-glycosylation sites in the protein which indicates that the ADAMTS1 gene product is a putative cysteine-rich secretory glycoprotein. Sequence analyses of porcine ADAMTS1 protein by ExPASy revealed that the porcine ADAMTS1 protein consists of one disintegrinlike domain,one zinc-dependent metalloproteinase and three thrombospondin typeⅠrepeat.3.Using the ImpRH panel,we determined that pig ADAMTS1 is closely linked with microsatellite marker S0215 on SSC13q49.4.Comparison of the sequences of ADAMTS1 in different pig breeds by using BLAST revealed two SNP.One of them was found within exon 7 of which a G-C substitution changes the codon for arginine into proline that spans a PvuⅡrestriction site.The other was found within intron 7 of which a G-A substitution that spans a StyⅠrestriction site. 5.The substitutions were situated within PvuⅡand StyⅠrecognition site and developed as PCR-RFLP markers for further use in population variation investigations and association analysis with litter size.Allele frequencies of this SNP were investigated in seven pig breeds/lines.An association analysis in new Qingping female line(116 new Qingping female line pigs;247 litter records) suggested that different ADAMTS1 genotypes have significant differences in litter size.6.RT-PCR results indicated that porcine ADAMTS1 gene is selectively induced in granulosa cells of preovulatory follicles by the hCG surge.Maximal levels of these proteases are observed at 12-16 h after an hCG surge,the time of ovulation.RT-PCR results indicated that porcine PR gene is induced in granulosa cells of preovulatory follicles by the hCG surge.Maximal levels of these proteases are observed at 8-12 h after an hCG surge,just before the ADAMTS1.Western blot results indicated that porcine PR protein is induced in granulosa cells of preovulatory follicles by the hCG surge.Maximal levels of these proteases are observed at 7-9 h after hCG surge.7.Genome walking technique was used to clone the proximal promoter region of porcine ADAMTS1.A fragment of 1458bp upstream start code was obtained from genomic DNA of porcine.Use computer analyze,tow PR site were found.Based on this fragment,together with different mutants of the proximal promoter cloned by PCR were cloned upstream of the EGFP reporter constructs and were analyzed using transfection and flow cytometry.The result showed that all 7 EGFP reporter constructs have no significant differences.8.Based on the porcine EST databases,a pair of primer was designed to amplify the DNA binding region of porcine PR gene.The DNA binding region of porcine PR gene was cloned into pcDNA3.1 vector.Co-transfection analysis was used to examine interaction between DNA binding region of porcine PR gene and ADAMTS1 promoter.The recombinant pcDNA-PR was co-transfected into COS-7 cells with ADAMTS1 promoter-EGFP.To confirm the interaction,three mutant ADAMTS1 promoter-EGFP for PR-binding site(mut) were also co-transfected with pcDNA-PR. Expression level in the cells were examined using flow cytometry.Electrophoratic mobility shift assay was employed to evaluate the combining activity of PR and ADAMTS1 promoter of granulosa cells.EMSA results indicated that the PR has binding activity on ADAMTS1 promer.The result showed that the PR plays important role in starting ADAMTS1 expression.