Screening Of MiRNAs Related To Egg Production Traits In Leizhou Black Duck Ovary Tissue And Verification Of MiR-34-x Function

This study takes Leizhou black ducks with high egg production and low egg production as the research object.Using transcript sequencing technology to conduct small RNA sequencing analysis on the ovarian tissues of high and low yielding Leizhou black ducks,screen for deferentially expressed miRNAs,and use GO and KEGG to predict the target genes of differential miRNAs related to egg production traits.Using q PCR,Edu,CCK 8 and flow cytometry methods,the effects of miR-34-x on the proliferation and apoptosis of follicular granulosa cells were studied.The target relationship between miRNA and target gene is verified by dual lucifer reporter gene detection technology,in order to screen out the regulation of egg production traits The role of miRNA and its target genes will lay the biological foundation for molecular breeding of egg production traits in Leizhou black ducks in the future.(1)In this study,the collected HG and LG Leizhou black duck ovarian tissues were subjected to small RNA sequencing.The results showed that the length of all small RNA sequences was up to 22 nt.A total of 29 miRNAs were differentially up-regulated and down-regulated between HG and LG groups.The results of the miRNA target gene KEGG pathway show that the signal pathways related to reproduction include Oxycontin,Gn RH,ovarian steroid production and PI3K/Akt signaling pathways;GO analysis showed that target genes were significantly enriched in biological processes,cell components,and molecular functions.Twelve deferentially expressed miRNAs were randomly selected.Using U6 as the internal reference gene,the accuracy of the sequencing results was verified by q PCR.As a result,the q PCR results are consistent with the trends of the sequencing results.The above research results enrich the database resources of duck miRNAs and lay the foundation for future research on miRNA regulation of ovarian development.(2)The primary follicular granulosa cells of Leizhou black duck were separated and cultured.The separation method was a combination of mechanical separation and enzymatic digestion,and the cells were digested with 0.1% type II collagenase.The semi-quantitative PCR was used to compare the expression of FSHR in ovarian and prefollicular granulosa cells of different grades,and the cell indirect immunofluorescence staining method was used to identify isolated granulosa cells.The above results indicate that the cells isolated and cultured in this experiment are follicular granulosa cells.(3)To explore the effects of miR-34-x on the proliferation and apoptosis of granulosa cells in Leizhou black duck follicles.By transfecting miR-34-x mimics and inhibitors in primary granular cells,q PCR,CCK8,Ed U,and flow cytometry were used to detect cell proliferation and apoptosis.CCK8 results showed that the mimics group was lower than the mimics NC group,and the inhibitor group was higher than the inhibitor NC group;Ed U test results showed that the proportion of Ed U-positive cells in the mimics transfected group was lower than that in the NC group,and the proportion of the transfected inhibitor group was higher than that in the NC group.q PCR detection of proliferation marker genes,the results showed that after transfection of mimics,the expression of CCND1 and CDK2 was extremely significantly reduced(P < 0.01);after transfection of inhibitor,CCND1 was extremely significantly increased(P < 0.01),and CDK2 was significantly increased(P < 0.05)).Flow cytometry showed that after transfection of mimics,the apoptosis rate increased,and the apoptosis rate decreased after transfection of inhibitor;q PCR detection of apoptosis marker genes,the results showed that after mimics transfection,the expression of Caspase3 and TP63 were significantly increased compared with the NC group(P < 0.05);and after the inhibitor was transfected,TP63 was extremely significantly reduced(P < 0.01);Based on the above results,miR-34-x can inhibit the proliferation of Leizhou black duck follicular granulosa cells and promote their apoptosis.(4)In order to study and identify candidate target genes of miR-34-x,PIK3 CG was obtained as a candidate target gene through bioinformatics methods.Use dual luciferase and q PCR for verification.Use dual luciferase and q PCR for verification.The results showed that the luciferase activity of PIK3 CG wild-type(WT)and miR-34-x mimics co-transfection group was significantly lower than that of the control group(P < 0.05),indicating that miR-34-x and PIK3 CG have a targeting relationship;q PCR showed that miR-34-x can target to inhibit the expression of PIK3 CG.Based on the above results,it can be concluded that PIK3 CG is a candidate target gene for miR-34-x.In summary,the results of small RNA sequencing found that there were 29 differentially expressed miRNAs in high-and low-yield Leizhou black ducks;miR-34-x can promote granulosa cell apoptosis,and PIK3 CG is the target gene of miR-34-x.