LncRNA Participating In The Metamorphosis Of Bombyx Mori Through MiRNA

Bombyx mori is an insect with complete metamorphosis,which has four stages of egg,larva,pupa and adult.Bombyx mori,as a model insect of Lepidoptera,plays an important role in the control of Lepidoptera pests.Development of Bombyx mori requires the participation of multiple hormones as well as ncRNAs.,studing the mechanosims of its developmentis of great importance in Bombyx mori industry and lepidopteran pest control.In the early stage of transcriptome sequencing of Bombyx mori,our research group found a long non-coding RNA TCONS₀0017454 is origin from the same genomic location of bmo-let-7、bmo-mir-100 and bmo-mir-2795 in the same strand.let-7 is very conservative in many species and plays an important role in determining the fate of cells.In order to study the biological function of this long non-coding RNA and its roles during Bombyx mori development.firstly,the expression profiles of TCONS₀0017454,bmo-let-7,bmo-mir-100 and bmo-mir-2795 in Bombyx mori at different developmental stages were constructed.Secondly,the effect of TCONS₀0017454 on the abnormal development of Bombyx mori was studied by injecting TCONS₀0017454 dsRNA into the individual Bombyx mori to interfere the expression of TCONS₀0017454.Finally,the molecular mechanism by which TCONS₀0017454 regulates Bombyx mori metamorphosis and development through let-7 miRNA clusters was studied by applying exogenous ecdysone 20 E in BmN cells.The main research results are as follows:1.Expression profile of TCONS₀0017454 and let-7 miRNA cluster in larval stage of Bombyx mori.The expression profiles of TCONS₀0017454 and let-7 miRNA cluster were detected by fluorescence quantitative PCR with the total RNA of silkworm larvae at different development stages as the research object.The results showed that the expression trend of TCONS₀0017454 was consistent with that of bmo-let-7 and bmo-mir-100,while there was no clear correlation between TCONS₀0017454 and bmo-mir-2795,indicating that the results suggest that miRNAs in the same gene cluster may have their own independent promoters.2.Expression profiles of TCONS₀0017454 and let-7 miRNA cluster in silkworm pupa stage and adult.The expression profiles of TCONS₀0017454 and let-7 miRNA cluster were detected by fluorescence quantitative PCR with total RNA at different developmental stages of pupal stage as the research object.The results showed that the expression trend of TCONS₀0017454 was consistent with that of bmo-let-7,but the correlation between TCONS₀0017454 and bmo-mir-2795 and bmo-mir-100 was not strong,indicating that there may be different interaction between lncRNA and miRNA in different stages of development of Bombyx mori.3.Interference with TCONS₀0017454 in vivo leads to abnormal development of Bombyx mori.by synthesizing dsRNA in vitro and then injecting it into Bombyx mori at the day 1of 3^rdd instar larvea.We observed that injection of TCONS₀0017454 dsRNA can causes Bombyx mori to not molt normally during different age transition.,diarrhoea and get smaller size of silkworm.Then the interference effect was tested,we found that after dsRNA injection,the relative expression levels of TCONS₀0017454 in silkworm were down-regulated by 80%,and the relative expression levels of bmo-let-7,bmo-mir-100 and bmo-mir-2795 were down-regulated by 40%,36%and 40%,respectively.4.The interaction between TCONS₀0017454 and let-7 miRNA was verified by cell experiments.BmN cells were treated with 5μM 20E for 48 h,RNA was extracted,and the expression of TCONS₀0017454 and let-7 miRNA cluster were detected by fluorescence quantitative PCR.The results showed that TCONS₀0017454,bmo-let-7,bmo-mir-100and bmo-mir-2795 were all upregulated by 20E.Then the target genes of bmo-let-7 were detected,and it was found that the relative expression levels of E74 and FTZF1 were significantly down-regulated,indicating that TCONS₀0017454 could regulate the expression of its target genes E74 and FTZF1 through miRNA let-7,thus participating in metamorphosis of Bombyx mori.