Functional Analysis Of MiRNA In The Ecdysone Biosynthesis Pathway Of Chilo Suppressalis

The rice stem borer Chilo suppressalis,is an important agricultural pest,causing huge losses on rice production.In this study,we cloned the 3’UTR of nine genes in 20E signaling pathway in C.suppressalis.Using five prediction softwares,we found several target sites on these 3’UTR.Then we used DLR vector to confirm the interactions between miRNAs and target 3’UTRs in vitro.qRT-PCR was used to detect the expression profiles of miRNAs in C.suppressalis metamorphosis process.The suitable injection time were determined by the expression profile analysis.Then the miRAN mimics were injected into C.suppressalis,to explore the function of miRNA in the metamorphosis process.1.3’UTR cloning of C.suppressalis 20E signaling pathway genesUsing RACE,we cloned the 3’UTR of nine C.suppressalis 20E signaling pathway genes,including csu-babo,csu-dib,csu-dsmad2,csu-erk,csu-nvd,csu-ptth,csu-punt,csu-ras,csu-shd.The average length of these 9 3’UTR is 526 bp,the longest 3’UTR is csu-ras(1043bp),and the shortest 3’UTR is csu-dib(55bp).2.Prediction of miRNAs’ targets on 20E signaling pathway genesWe used five softwares to predict the targets of miRNA on 20E signaling pathway genes and screened the targets which were predicted by four or five softwares.As results showed,csu-nvd gene is the target of csu-miR-9b,csu-miR-239,csu-miR-241;csu-spook gene is the target of csu-miR-bantam,csu-miR-024,csu_miR-80,csu-miR-89,csu-miR-124,csu-miR-154,csu-miR-257;csu-dib gene is the target of csu-miR-260.3.In vitro target validation between 20E signaling pathway genes and miRNAsWe used DLR system to confirm the interactions between 10 miRNAs(csu-miR-8,csu-miR-9b,csu-miR-bantam,csu-miR-80,csu-miR-89,csu-miR-124,csu-miR-154,csu-miR-241,csu-miR-257,csu-miR-260)and 20E signaling pathway genes(csu-nvd,csu-dib,csu-spook).Experiment results showed that,csu-nvd gene is the target of csu-miR-9b and csu-miR-241;csu-dib gene is the target ofcsu-miR-260;csu-spook gene is the target of csu-miR-bantam,csu-miR-80,csu-miR-89,csu-miR-154,csu-miR-257.4.Daily expression profiles of 7 miRNAs in the metamorphosis process of C.su ppressalis.Using qRT-PCR,we got the daily expression profiles of seven miRNAs in the metamorphosis process of C.suppressalis.Results showed that,csu-miR-9b has high expression during 5 instar larva;csu-miR-bantam,csu-miR-80 has high expression during 6th instar larva;and csu-miR-89,csu-miR-154,csu-miR-257,csu-miR-260 has high expression during pupa stage.We selected the 4th day at the 6th instar to inject miRNA mimics.5.Functional analysis of miRNA in the metamorphosis of C.suppressalis.We synthesized the mimics of csu-miR-9b,csu-miR-bantam,csu-miR-80,csu-miR-89,csu-miR-154,csu-miR-257,csu-miR-260,to explore the function of these miRNAs in 20E signaling pathway of C.suppressalis.The high death rate and pupation delay were observed in all three mimics-treated groups.Three mimics-injected groups showed lower pupation,higher mortality,and lower 20E titer.The results showed that in the metamorphosis process,csu-miR-9b suppress the 20E signaling pathway by targeting csu-nvd,csu-miR-bantam,csu-miR-80,csu-miR-89,csu-miR-154;csu-miR-257 targets on the csu-spook gene;csu-miR-260 targets on the csu-dib gene,leading to the deceasement of 20E titer,which arrest the metamorphosis of C.suppressalis.