Cloning, expression and target gene prediction of let-7 in Paralichthys olivaceus metamorphosis

Abstract

The let-7 gene is a critical regulator of developmental timing events of the larval-to-adult transition.Here,Paralichthys olivaceus let-7 was identified by a stem-loop RT-PCR.The precursor of let-7 was cloned and was proven to be highly conserved among different species.Quantitative analysis revealed that the expression levels of let-7 were generally variably enhanced during the first five metamorphic stages and peaked during the late metamorphic stages(36 dph).At the end of P.olivaceus metamorphosis(41 dph),expression of let-7 was re-duced.To study the effects of triiodothyronine(T3) on the expression of let-7,six different concentrations(0 nmol?L?1,50 nmol?L?1,75 nmol?L?1,100 nmol?L?1,200 nmol?L?1) were used to treat P.olivaceus kidney cells in vitro.Quantitative PCR showed that let-7 levels increased after T3 treatment,especially at 75 nmol?L?1,indicating that T3 regulated the expression of let-7.Further investigation of target genes demonstrated that the 3′-untranslated regions(3?-UTRs) of 29 mRNAs were possible target sites matched with let-7.These mRNA targets played key roles in diverse biological processes: development,cell proliferation,signal transduction,virus immune response,and signaling pathways.These re sults suggested that let-7 played an essential role in regulating the developmental timing of P.olivaceus metamorphosis.