Roles Of MiR-286 In Reproductive Development In Bactrocera Dorsalis Females

Female reproductive potential is crucial for a population to multiply.Micro RNAs(miRNAs)have been demonstrated to be critical regulators in the post-transcriptional regulation of numerous biological processes,including reproduction.However,these studies mainly focus on model species.The oriental fruit fly,Bactrocera dorsalis,is a highly invasive pest.Therefore,the identification of miRNAs involved in reproductive development in B.dorsalis not only can enrich and improve the mechanism of insect reproductive regulation,but also provide new targets and ideas for the sustainable management of B.dorsalis.We have established eight micro RNA libraries of B.dorsalis and screened out three known miRNAs that are differential expressed in high abundance in female adults.Their expression profiles were detected by q PCR at different stages of reproductive development of female adult fat body and ovary tissues.The results showed that miR-286 was specifically high expressed in the ovary and fat body,and the expression level was significantly increased during the development.The miR-286 antagomir was injected into the female adults by micro-injection techniques to detect its function in reproductive development.The molecular mechanism by which miR-286 regulates the reproductive development of female adults of B.dorsalis was clarified,using target gene prediction,dual luciferase verification,RNAi,rescue experiment,etc.1.Identification of differentially expressed miRNAs and their spatiotemporal expression pattern during reproductive development of B.?dorsalis.Eight miRNA libraries were established in the ovary and fat body from four developmental stages [1 days(d),5 d,9 d,and 13 d post-eclosion].116 and 84 miRNAs were found to be differentially expressed in ovary and fat body,respectively,during sexual maturation of female adults.The Gene Ontology analysis of the target genes involved in oogenesis and lipid particle accounted for 33% and 15% of the total targets,respectively.Among these differentially expressed miRNAs,there are 25 miRNAs with high abundance difference in both ovary and fat body.Three highly differential expressed miRNAs(miR-286,miR-317-3p,and miR-2779)were selected for q PCR verification that are shared in the ovary and fat body.The results are basically consistent with the sequencing data.The expression levels of miR-317-3p and miR-2779 were significantly up-regulated during sexual maturity of fat body;miR-286 was significantly up-regulated during fat bodiy and ovary during sexual maturity.Tissue differential expression patterns show that miR-286 is highly expressed in ovary and fat body.2.Function alanalysis of miR-286 in reproductive development.The miR-286 inhibitor(Antagomir)was injected into female adults of B.dorsalis by micro-injection technology,and its function in reproductive development was tested.The results showed that after injection of Ant-286,ovarian development was delayed,the average daily egg production and total egg production were significantly reduced.There was no significant change in hatchability.After further detection by q PCR,it was found that inhibition of miR-286 expression resulted in significant down-regulation of the vitellogenin(Vg)Vg1,Vg2,and vitellogenin receptor gene Vg R.The above results indicate that miR-286 may affect its reproduction by affecting the yolk deposition,ovary development and fertility of B.dorsalis females.3.Prediction,verification and functional study of miR-286 target gene.Through Target Scan and miRanda software to predict miR-286 target genes,select the intersection of the two software predictions and obtain a total of 16 potential target genes.Using q PCR to detect the effect of potential target genes expression after injection of miR-286 inhibitors in female B.dorsalis.The results showed that the expression level of 9 potential target genes was down-regulated after injection of Ant-286.The expression level of 1 potential target gene was down-regulated after injection of Ant-286.Further,the expression patterns of 10 potential target genes in different developmental stages of female adults were detected by q PCR,and it was found that the expression patterns of genes uncharacterized protein LOC105232785,dual specificity mitogen-activated protein kinase hemipterous-like,protein escargot and miR-286 were opposite in different periods.Next,the dual luciferase report experiment showed that UCP is a potential target gene of miR-286.In order to further clarify the function of UCP,we used RNAi technology to silence UCP in female adult.The results showed that ovarian development was inhibited,and the egg production was significantly reduced.The injection of ds UCP had no significant effect on hatching rate.After q PCR detection,the expression levels of Vg1,Vg2 and Vg R were significantly up-regulated after UCP was silenced,and Endophilin B1 was significantly down-regulated.The above results indicate that after silencing UCP,Vg cannot be absorbed by the ovary due to endocytosis,and the ovarian development of B.dorsalis is blocked,and the fertility is reduced.4.ds UCP and Ant-286 co-injection for rescue experiment.In order to clarify the regulation mode of miR-286 targeting gene UCP and the regulation of reproductive development,the mixture of ds Egfp/Ant-286,ds UCP/Ant-286 and ds UCP/Ant-NC was injected into the female adults of B.dorsalis by microinjection,and their functions in reproductive development were examined.The results showed that compared with ds Egfp/Ant-286,the ds UCP/Ant-286 rescued the delayed ovarian development and decreased spawning.Furthermore,the expression levels of Vg1,Vg2,Vg R and Endophilin B1 of B.dorsalis were detected by q PCR.It was found that the expression levels of Vg1,Vg2,Vg R and Endophilin B1 in the ds UCP/Ant-286 were significantly increased compared with the ds Egfp/Ant-286,and there was a significant rescue effect.The above results indicate that UCP silencing can rescue the effects of miR-286 inhibitors on the yolk deposition and transport and eggs laid of female B.dorsalis.In summary,miR-286 regulates the reproductive development of B.dorsalis by regulating the expression of the target gene UCP.This research can not only enrich and perfect the regulation mechanism of insect reproductive development,but also lay a theoretical foundation for the green prevention and control strategy of B.dorsalis.