The Effects Of BMP15 Gene On Proliferation And Differentiation Of Cashmeregoats MGSCs

BMP 15(Bone morphogenetic protein 15)is a member of transforming growth factor ?(TGF?)superfamily.In early,BMP15 is considered only specific expression in the oocyte,and have important regulatory roles in the fecundity of female.But with the deepening of the research,BMP15 expression was found in pituitary,hypothalamus,testis and other tissues in some species,suggesting that BMP15 may also regulate the development of other tissues.In this study,the bioinformatics method was used to analyze the cDNA sequence information of goat BMP15 and predict its coding protein in the physical and chemical properties,secondary structure,signal peptide and tertiary structure,etc.,in order to further study the functional adjustment mechanismof BMP15 proteinto lay the theoretical basis.In goat mGSCs culture in vitro,the overexpression of BMP15 gene,the role ofBMP15 in mGSCs proliferation and differentiation could be preliminarily explored.These results could provide reference for exploring BMP15 role in the development of testis.In this study the main results were as follows:1.The mGSCs of Shanbei white cashmere goat isolated and purified by hybrid enzyme digestion and differential attachment method.Differential attachment culturing after7 d,by the AKP staining,the positive clone accounted for about 60%.After immunohistochemical staining of CD49 f and Oct4,it showed that the expression of CD49 f and Oct4 were larger,and mGSCs obtained were pure.Embryonic bodies(EBs)were found by hanging drop to cultivate undifferentiated mGSCs.The osteoblasts,chondrocytes and adipocytes were found to be cultured after 7 days of EBs adherent culture.The mGSCs of Shaanxi white cashmere goats were identified to have good differentiation potential.These results showed that Shanbei white cashmere goat mGSCs by culturing in vitrohave better differentiation potential.2.Using bioinformatics analysis software,the structure and function of goat BMP15 gene sequence and its coding protein were analyzed.It found that the size of goat BMP15 protein coding region is 1185 bp,encoding 394 amino acids,and its gene sequence and protein sequence homology with sheep and cattle were 99 % and 98 %,respectively,the genetic relationship being close.According to the analysis of signal peptide,we predicted that the best shear site may exist between 25 and 26 amino acids,and D value is 0.815,suggesting that goat BMP15 protein may be a growth factor and belong to secretory protein,havingsignal transduction function.Using Smart online program to predict and analyse BMP15 protein functionandstructure domain,it showed that BMP15 have TGF? functiondomain,located at 293-394 amino acids region.Using ProtFun online program to predict and analyse BMP15 protein function,it showed that goat BMP15 has themostpossibility inthe function of cell envelope,and odds reach 12.822,and speculated that after synthesis,BMP15 may be transported to the cellenvelopeto do functional response.By analyzing gene ontology category,we found that BMP15 as a hormone to pass information in physiological activity have the most possibility.3.The expression of bmp15 in different tissues of adult cashmere goats detected byRT-PCR,a certain expression of BMP15 was found in testis,duodenum,rumenand other tissues,speculating that BMP15 have a regulatory role in the physiological processes of these tissus.After the overexpressionof BMP15 in mGSCs,the resultsshowed thatthe expression of c-Kit was increased,and the expression of PLZF was decreased,but both no significant difference with the control group(P> 0.05),so it was speculated that BMP15 had a certain influence on the proliferation and differentiation of mGSCs.In this study,the results showed that cashmere the cultured goat mGSCs in vitro had good potential for multi-directional differentiation;BMP15 have TGF? functiondomain and the function of signal transduction,and have a certain influence on the proliferation and differentiation of mGSCs.