MicroRNA Cluster MiR-2?13?71 Regulates Vitellogenesis And Oocyte Maturation By Targeting Notch In The Migratory Locust

The migratory locust is a representative of hemimetabolous insects.It is also a worldwide destructive insect pest with high fecundity.The female reproduction of migratory locusts is mainly regulated by juvenile hormone(JH).JH promotes vitellogenin(Vg)expression in the fat body,secretion into the hemolymph,transportation to the ovary,and deposition into developing oocytes.MicroRNA(miRNA),a class of small noncoding RNAs in animals,plants,fungi and viruses,regulates the gene expression at post-transcriptional level through translational repression or mRNA degradation.Our previous studies have shown that the expression of miRNAs is regulated by JH in locusts.By knockdown of Ago1,we have demonstrated that Ago1-dependent miRNA pathway is indispensable to locust vitellogenesis,ovarian development and oocyte maturation.However,the molecular basis of miRNA function and regulation in insect reproduction remains unclear.In this dissertation study,by using approaches of omics,bioinformatics and molecular biology,we show that miR-2 cluster(miR-2?13?71)regulates locust vitellogenesis and oocyte maturation through its target gene Notch in the fat body.The miRNA transcriptome of adult female fat body was obtained through the illumina high throughput sequencing.Together with the locust genome database,we identified 25 miRNA clusters in the genome.Five miRNA clusters with the length <1500 nt were selected for further analysis.Among them,two clusters showed constitution of linear conserved miRNAs.We found that the expression of miR-2 cluster gradually decreased after adult eclosion.miR-2 cluster comprised of miR-2,miR-13 and miR-71 was located in scaffold29860.qRT-PCR revealed that miR-2 cluster was significantly decreased post JH analog(JHA)treatment.To explore the functions of miR-2?13?71 in locust vitellogenesis and oocyte maturation,we overexpressed the miR-2?13?71 by injection of miRNA agonist(AgomiR).The data showed that overexpression of miR-2?13?71 led to blocked ovarian development and arrested oocyte maturation.Statistically,the average length of primary oocyte was significantly shorter than that of the control group.Subsequent qRT-PCR demonstrated that the transcription levels of VgA and VgB are significantly reduced the fat body after injection of miR-2 cluster AgomiR.Taken together,the above results indicate that overexpression of miR-2?13?71 suppresses the expression of Vg,consequently leading to inhibited ovarian development and oocyte maturation.To unveil the mechanisms of miR-2?13?71,we used locust genome database and miRanda software along with miRNA transcriptome for a large scale of screening to identify the targets of miR-2 cluster.After the predicted miRNA targets were annotated,Notch pathway was significantly enriched in the KEGG analysis.Dual-luciferase reporter assay and RNA co-immunoprecipitation demonstrated that Notch was the target of miR-2?13?71,and overexpression of miR-2?13?71 reduced the expression of Notch.Depletion of Notch via RNAi resulted in decreased levels of Vg mRNA,blocked ovarian development and arrested oocyte maturation,resembling the defective phenotypes caused by the overexpression of miR-2?13?71.Notably,the transcript levels of Notch was significantly enhance by JH treatment.In conclusions,the results from this dissertation research indicate that miR-2?13?71 regulates locust vitellogenesis and oocyte maturation via targeting Notch gene.Though miR-2?13?71 targets Notch,JH upregulates the expression of Notch and simultaneously downregulates the expression of miR-2?13?71.This mechanism ensures the precise control of Notch expression required for JH-dependent vitellogenesis,oocyte maturation and egg production.