| The red flour beetle,Tribolium castaneum is a typical holometabol.an insect,it is lifecycle go though different stages including embryo,larval,pupal and adult.As the morphological characteristics and functions were diverse in different stages,indicating the biological process of its metamorphosis are complex.In holometabolan modes,metamorphosis is basically regulated by two hormones,the molting hormone(generally 20-hydroxyecdysone)and the juvenile hormone(JH).However,the holometabolous development mechanism in metamorphosis still largely uncleared.Previous studies have figured out that a small non-coding RNAs,called microRNAs,palyed important roles in organism development.As a class of key regulators,miRNAs were found closely related to insect metamorphosis.Here,we used the T.castaneum as a model,selected 5 miRNAs that specific lowly expressed in early pupa stage,increased miRNA expression level by injected miRNA mimic,further investigated its function in larvae development and metamorphosis.The results were as follows:First,injected specific mimics of tca-miR-8b-3p,tca-miR-8c-3p,tca-miR-9a-5p,tca-miR-252c-5p,tca-miR-3017b about 200uM each individuals into fifth instar larvae were carried on for further explored its function in vivo The results displayed that tca-miR-8b-3p,tca-miR-8c-3p,tca-miR-9a-5p,tca-miR-252c-5p mimics treatments showed no significantly difference between controls.However,tca-miR-3 017b mimics treatments exhibited significantly defects to controls,the phenotypes showed prevented moulting from larvae to pupae transition and caused lethal effects.Besides,the molting from larva into pupa stage were delayed approximately 3-4 days.Second,in order to further explore tca-miR-3017b regulation networks during metamorphosis and molting,three algorithms including miRanda,PicTar and Targetscan were carried on for target prediction.At last,Several genes(csnk-1,ARHGDIB,actin,serca,GCP60,PDK,BCL2)were sceened out in overlapped results.We futher constructed Reporter plasmid by inserted its CDS or 3'UTR sequences which containe tea-miR-3017b functional sites.Thus,tca-miR-3017b and its candidate targets validation were carried on in vitro.The results showed that co-transfected with miR-3017b mimics and target sites plasmids reduced 60%luciferase activity than blank control,which indicating that serca was functional target of tca-miR-3017b.Finally,To further investigate tca-miR-3017b amd its target serca regulating T.castaneum development,the function of serca,injection of dsRNA of Tcserca into last-instar larvae led similar phenotype with injection tca-miR-3017b mimics,larvae cannot molting normal pupa,and then larvae dark dry death,and this phenotyoe is 100%,further demonstrate that serca was the target gene for miR-3017b.The results showed that miR-3017b and its target gene serca were involved in the molting and metamorphosis,and we examined the key genes of ecdysone and juvenile hormone pathway,using qRT-PCR,we evaluated the transcriptional level of E74,BR-C,Ftz-f1l,CYP18A1,Kr-h1,Met in larvae with the tca-miR-3017b mimic injection and dsserca injection.Injection of dsserca into last-instar larvae led similar with injection tca-miR-3017b mimics,the transcriptional level of CYP18A1 gene in the whole body of T.castaneum was significantly reduced as compared to control.The CYP18A1 gene is a key enzyme that inactivates 26-hydroxylation of 20E.In conclusion,these findings showed that miR-3017b is involved in regulating T.castaneum larval development by directly targeting serca and indirectly regulate CYP18A1 expression level led to successful molting.Moreover,this study demonstrate that spatiotemporal control of miRNA expression is crucial for the successful completion of insect development.Using RNAi to clarify the function of serca.At the application level,the lethal effect of miRNA mimetic injection indicates the application value of miRNA in pest control,which provides the theoretical and practical basis for miRNA as a potential new type of insecticide. |
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