The Expression Of Chicken MiR-199a In The Gonads Of Chicken Embryos And The Construction Of Its Overexpression System

The development of chicken embryo gonads shows dimorphic that the development of female and male chicken embryo gonads is not the same.Male chicken embryos have bilateral gonads developing into testes,while female chicken embryos have degenerated gonads on the right side,and the left gonads develop into ovaries.This biological process is the result of many biological factors,but its specific mechanism has not been reported yet.miR-199 a is a miRNA that can regulate cell proliferation and apoptosis.However,there are not many studies on the role of gga-miR-199 a in chicken embryo gonadal development.One of important reason is that few cell lines can be used to study chicken gene functions in vitro.In addition,chicken cells culture has relatively high requirements for temperature and serum,so it is not easy to carry out related research.To explore the role of gga-miR-199 a in chicken gonadal development,this study isolated chicken embryo gonads of different ages and detected the expression of gga-miR-199 a.At the same time,a lentiviral system overexpressing gga-miR-199 a was constructed as a basic tool to study the effect of gga-miR-199 a in chicken cell lines,primary cells,and in vivo.The results of the study are as follows:1.To explore the role of gga-miR-199 a in the chick embryo gonads,this study separated the left and right gonads of different ages of two gender chicken embryos.The expression of gga-miR-199 a and its target genes were detected by RT-q PCR.The results showed that the proliferation or degeneration of the right gonads of male and female embryos may be related to the expression of gga-miR-199 a,and the development-related genes HIF1 and Smad1 may be involved in the development of chicken gonads as the target genes of gga-miR-199 a.These results indicated that gga-miR-199 a plays a certain role in the development of chicken gonads.2.A lentiviral expression system overexpressing gga-miR-199 a was successfully constructed.A HEK293 T cell line that overexpresses gga-miR-199 a stably and continuously was obtained by infecting the system.The expression of gga-miR-199 a of overexpression cells was higher 191.76 folds than normal cells.There are 699 differentially expressed genes were screened between overexpression and normal cells by transcriptome sequencing.These differential expression genes were enriched 34 biological processes and 13 related signals path by GO and KEGG into.These results show that the overexpression of gga-miR-199 a can affect the biological process of the cells.3.Using the lentiviral expression system,gga-miR-199 a was overexpressed in chicken DF-1 cell lines and embryo primary cells,and DF-1 cell lines that overexpress gga-miR-199 a stably and continuously were obtained.The expression of gga-miR-199 a measured by RT-q PCR in DF-1 cell line was extremely significantly increased and the expression of the target gene HIF1α was significantly reduced by the lentiviral expression system.In the chicken embryo primary cells,the expression of gga-miR-199 a and its target gene HIF1α had a tendency to increase and decrease after treating by the lentiviral expression system.In summary,gga-miR-199 a plays a regulatory role in the development of chicken embryo gonads by regulating target genes related to growth and development.The specific mechanism of the regulation needs further study.In this study,the expression of gga-miR-199 a in DF-1 cells and chicken embryo primary cells were overexpressed by constructing and transfecting the lentiviral expression system.The lentiviral expression system provided a basic tool for studying the function of gga-miR-199 a in chicken gonadal development.