Cloning,expression profile and ovarian localization of sheep matrix Gla protein gene
The aim of this study was to clone the protein coding sequence(CDS)of sheep matrix Gla protein(MGP)gene to prepare specific antibody and to detect the expression distribution of the gene in sheep o-varian tissues.Total sheep follicle cDNA was used as the template to amplify and obtain the full-length sequence of the CDS region of sheep MGP gene.Peptide with a length of 15 amino acids from the C-terminal end of the sheep MGP protein was synthesized and was used to immunize mouse and perpare antiserum.Expression levels of MGP in different tissues and follicles of varying sizes were analyzed by real-time flu-orescence quantitative PCR(qPCR)and immunoblotting technology.Immunohistochemistry method was used to analyze the expression and distribution characteristics of MGP in ovarian tissue.The results revealed that the overall fragment(467 bp)of sheep MGP gene CDS region was obtained successfully,and the fragment encoded 103 amino acids,with a theoretical relative molecular weight of 12 230.96 and an isoelectric point of 9.27.Sequence composed of 19 amino acids from the N-terminal was predicted as the signal peptide sequence,and the mature peptide contained 84 amino acids,which was extra-membranous domain protein without intracellular region.The total protein amino acid sequences of MGP shared similarities with those of the homologous proteins from goats and cows of 100.0%and 99.0%respectively,and the similarity with hu-man and mouse was 85.4%.The relative expression levels of MGP mRNA and protein were low in sheep tissues such as heart,liver,spleen,lung and kidney,but were high in reproductive systems or tissues,such as ovary,fallopian tube and uterus,and the relative expression level was also high in corpus luteum.The expression levels of MGP granulosa cells and cumulus cells were high,whereas it didn't expressed in atresia follicles.The study result can provide a basis for further in-vestigation of the biological role of MGP in ovarian tissues.
万方数据
维普
