| Paralichthys olivaceus,as one of the high-quality Marine economic fish species in China,is widely farmed in northern China.Its meat is delicious with high protein content and has high nutritional value.In the process of factory farming,it was found that the female fish is large and its growth rate is significantly higher than that of the male fish.In sex determination and sex gland differentiation and development process,the role of somatic Cell become more and more scholars studied seriously,somatic cells in embryonic period not only promote the primitive Germ cells(Primordial Germ Cell,PGC)with differentiated into bisexual potential reproductive primordium,related gene expression in cells of sex determination,also played a crucial role,after sexual maturity,gonad in not only provide Germ Cell proliferation and differentiation of places and nutrition,but also can secrete estrogen or androgen promote proliferation and differentiation of Germ cells.Therefore,it can be said that somatic cells are closely related to all the functions of gonads from the embryonic stage.However,there are few reports on the regulation of somatic cells on germ cells in bony fish,especially the role of miRNA in it.This field has become one of the hot spots of fish developmental research in recent years.According to the high-throughput sequencing results of miRNAs in the female gonads of Japanese flounder in our previous laboratory,a large number of miRNAs were differentially expressed in the testis and ovaries of male and female Japanese flounder.On this basis,we selected 4 families of miR-200 whose members were differentially expressed in testis and ovary,namely miR-200 a,miR-200 b,miR-141,and miR-429.On this basis,bioinformatics method was used to predict whether a group of sex-related genes(amh、foxl2、dmrt1、wnt4、sox9、gsdf)had possible binding sites with this family.The results showed that only amh had binding sites with this family,and due to the strong conservatism of miR-200 family,two possible binding sites were predicted in the 3’UTR region of amh.Therefore,in this study,the 3’UTR region of amh was first constructed into the dual luciferase reporter vector,and the targeting relationship between the two was ve rified in HEK293 T cells.Preliminary results showed that among the four members,miR-200 a,miR-200 b and miR-141 had a targeting relationship with amh,among which miR-200 a and miR-200 b had the most obvious inhibitory effect on amh.For clear miR-200 a and miR-200 b by any site to regulate the expression of amh,in the building in front of the double luciferase report carrier for fixed point mutation technology,may be two possible sites for bases,unrelated results further displayed on the site after a mutation,the original miR-200 a and miR-200 b inhibition of amh was canceled,and the locus mutation after two still exist.Consistent with dual luciferase report system,by building a green fluorescent protein carrier report found that cloned into amh 3 ’UTR green fluorescent protein in miR-200 a,miR-200 b under the action of green fluorescent protein decreased significantly,and when the site after a mutation green fluorescent protein expression and normal,and the locus mutation two green fluorescent protein is reduced.These results fully demonstrated that miR-200 a and miR-200 b regulated the expression of amh through site one,indicating that amh was a direct target gene of miR-200 a and miR-200 b.In addition,it was found that miR-200 a significantly regulated the 3’UTR of amh in all of these reporting systems.To the collapse of the cells of reproductive cells,we cultured cells were the gonads,and separate the somatic cells and reproductive cells,to adopt the method of fluorescence quantitative PCR first identified some gender related genes expression in testis germ cell and sertoli cells in the differences and gender related gene expression in ovarian granulosa cells.The results showed that,except for the vasa germ cell marker gene,the other gender-related genes such as cyp19a1、dmrt1、gsdf、sox9、amh were mainly expressed in the supporting cells or granulosa cells.To further understand how miR-200 a regulates the role of amh in the testis,we transfected miR-200 a mimics in the supporting cells of flalichthys olivus.The results showed that compared with the NC group,the expression of miR-200 a was significantly increased,while the expression of amh was significantly decreased.At the same time,the expression levels of male sex-related genes dmrt1 and gsdf also increased significantly,but dmrt1 and gsdf were not the target genes of miR-200 a.These results suggested that miR-200 a could inhibit the expression of amh in testis supporting cells and participate in gonad development and gamete formation in testis,while amh could negatively regulate the expression of male-related genes.In conclusion,the study in this paper lays an important foundation for the functional role of AMH-mediated miR-200 family in the gonad. |
PDF Full Text Request