The Regulation Of MicroRNAs In Molting Of Nymphs Of Arma Chinensis:Functional Analysis Of MiR-92a

Biological control is an important method of integrated pest management.Arma chinensis is a dominant natural enemy,which preys on a variety of agricultural and forestry pests such as Lepidoptera,Coleoptera and Hymenoptera,and plays a role in biological control.However,the growth and development period of nymphs of A.chinensis is long in artificial rearing process,and propagation of this natural enemy consume much time and laborious.Mi RNA as a small non-coding RNA,is involved in many biological processes of insect.Currently,the role of miRNA in the growth and development of A.chinensis is still unclear.In this study,high-throughput sequencing was conducted for nymphs of A.chinensis,and bioinformatics analysis was conducted based on the genomic information of A.chinensis,expression profiles were analyzed for partial miRNA,and miRNA-m RNA interactions were verified in vitro and vivo,providing a theoretical basis for revealing the regulate mechanism of miRNA in the molting of A.chinensis.The main results as follows:1.In this study,fifteen small RNA libraries were constructed for five different instars of nymphs of A.chinensis,and a total of 46 conserved miRNAs and 18 novel miRNAs were identified by high-throughput sequencing and bioinformatics analysis.Differential expression analysis of miRNAs in two adjacent instars of nymphs showed that specific miRNAs were up-regulated and down-regulated in different instars.RNAhybrid,miRanda and PITA were comprehensively used to predict target genes of significantly differentially expressed miRNA,and a total of 2913 target genes were predicted.The results of GO annotation analysis and KEGG pathway enrichment analysis of target genes indicated that miRNAs were mainly involved in biological regulation and immune pathways.2.In this study,the sequence of ecdysone receptor was determined based on the genome of A.chinensis,then analyzed the miRNA which targets to ecdysone receptor combining with sequencing results,and three conserved miRNAs were obtained:miR-9b,miR-92a,and miR-1180.The expression patterns of three miRNA and ecdysone receptor in the fourth instar nymphs after molting for one day to the fifth instar nymphs after molting for five days were quantified by real-time polymerase chain reaction（RT-PCR）.The results showed that the ecdysone receptor was highly expressed at the late stage of the 4^thand 5^th instar nymphs,but the expression was decreased at the initial stage.The expression of miR-92a was low at the end of the fourth and fifth instar nymphs,and increased at the preliminary stage.The other two miRNAs did not show a completely opposite expression pattern to the ecdysone receptor.3.In this study,the interactions between three miRNAs and ecdysone receptor were verified by dual luciferase reporter assays.The results showed that three miRNAs targeted to ecdysone receptor,among which miR-92a significantly suppressed transcription of ecdysone receptor,indicating targeting relationship is the strongest.Mi R-92a was further overexpressed,and RNA was extracted after 24 hours for quantitative real-time PCR,the expression of ecdysone receptor was down-regulated.Phenotypic observation showed that the molting rate of A.chinensis decreased and the mortality increased after the injection of miR-92a mimics.