Let-7a And MiR-278 Regulate Vitellogenesis And Oocyte Maturation By Targeting Krüppel-homolog 1 In Locusta Migratoria

The migratory locust,Locusta migratoria is a destructive insect pest in agriculture,with high fecundity.The reproduction of migratory locust is mainly regulated by juvenile hormone(JH).JH promotes vitellogenin(Vg)synthesis in the fat body,release into the hemolymph,transportation to the ovary,and uptake by the developing oocytes through patency in the follicular epithelium and receptor-mediated endocytosis at the oocyte membrane.Previous studies have shown that JH regulates the transcription of its early response gene Krüppel-homolog 1(Kr-h1)through the receptor complex Met/Taiman to promote locust vitellogenesis and egg production.Therefore,Kr-h1 is a key mediator in locust reproduction.microRNA(miRNA),a class of non-coding single stranded RNA with a length about 23 nucleotides,plays important roles in insect development,metamorphosis and reproduction.miRNAs bind to Argonaute(Ago)to form an RNA-induced silencing complex(RISC),consequently regulating gene expression at the post-transcriptional level by translational inhibition or mRNA degradation.Previous studies have demonstrated that the expression of miRNA in the fat body of adult female locusts is regulated by JH.Depletion of Ago1 via RNAi severely inhibits ovarian development and egg production,indicating the pivotal roles of miRNAs in locust vitellogenesis,oocyte maturation and egg production.However,the molecular mechanisms of miRNA regulation in insect reproduction remain poorly understood.By using the migratory locust as a model system,this dissertation study was set out to identify the miRNAs that regulates Kr-h1 and elucidate the underlying mechanisms.Using miRNA sequencing and quantification with small RNA libraries derived from fat bodies of female adult locusts,we identified 60 conserved miRNAs,followed by the predication of candidate miRNAs and their binding sites to Kr-h1 with the miRanda-3.3a software.Luciferase assays and RNA Immunoprecipitation(RIP)documented that let-7a and miR-278 bound to Kr-h1 CDS.qRT-PCR was employed to characterize the developmental profiles of let-7a and miR-278 in the fat body as well as the effect of JH treatment on the expression of Let-7a and miR-278.The data documented that miR-278 and let-7a expression was suppressed by JH.Overexpression of let-7a and miR-278 by injection of miRNA agonist(agomiR)resulted in significant decrease of Kr-h1 expression in the fat body,accompanied by significantly reduced levels of Vg,arrested ovarian development and blocked oocyte maturation.Based on the above findings,we propose that let-7a and miR-278 are down-regulated by JH,which maintains the normal levels of Kr-h1 to transduce the JH signaling during locust vitellogenesis and egg production.When let-7a and miR-278 are abnormally overexpressed,Kr-h1 expression is inhibited,which leads to significant decline of Vg transcripts as well as arrested ovarian development and oocyte maturation.Collectively,the data indicate that let-7a and miR-278 regulate vitellogenesis and oocyte maturation by targeting Kr-h1 in locusts.Our research thus reveals the functions of miRNA by cross-talking with Kr-h1,which advances the understanding of the epigenetic regulation in JH signaling pathway.