Identification Of Non-coding RNA In Five Insect Species And Functional Analysis Of MiR-14

Non-coding RNAs are the RNA molecules which do not produce protein.Among a variety of non-coding RNAs,miRNA and IncRNA have been shown to have important biological functions.Metamorphosis is a typical development characteristic of insects.In this work,we constructed a bioinformatics pipeline to predict non-coding RNA in five insect species.According to the miRNA gene family analysis,we found the insect-specific miRNA gene miR-14.Multiple sequence alignment indicated the high sequence conservation for miR-14 among insect species.Target prediction and validation,expression profile analysis,over-expression and inhibit expression of miR-14 showed that miR-14 targeted 9 genes in the ecdysone biosynthesis and response pathway.All the results indicated that miR-14 was important for maintain the stability and consistency in silkworm development.The main works are as follows:1.Identification and characteritic analysis of noncoding RNA in five insect species.We sequenced the small RNA library of Tryporyza incertulas,Spodoptera exigua,Plutella xyllostella,Laodelphax striatellus,and N.lugens.We constructed a bioinformatics pipeline to predict miRNA gene in these five insects.The predicted miRNA numbers for the five species were 76,64,108,118 and 110,respectively.We constructed a bioinformatics pipeline to predict the IncRNA gene in 12 transcriptome of brown planthoppers(BPH),N.lugens.The twelve samples were as follows:adult-2d low-fecundity femal,adult-2d high-fecundity femal,5th instar low-fecundity femal,5th instar high-fecundity femal,Mudgo fatbody,Taichung Native 1 fatbody,Mudgo salivary gland,Taichung Native 1 salivary gland,Izumo87 strain,Chikugo89 strain and a normal population.After filtering by Blastx,ORF length,CPC score,Pfam domain and Rfam analysis,we found 1,882 lncRNA genes.LncRNA in BPH shared the same characters with human,worms and zebrafish.In contrast to protein-coding genes,IncRNAs showed a bias of two-exon transcripts.The transcript length of lncRNA was significantly shorter than protein-coding gene(841 bp vs 1106 bp,T-test,P<0.01)and about 19%of the lncRNA gene were alternative spliced.Expression cluster analysis showed that most lncRNAs were population-specific expressed.Three IncRNA genes were found overlap with three fecundity-related protein-coding gene which suggest that IncRNA might involve in the regulation of BPH fecundity.2.miRNA gene family analysis showed that miR-14 was insect-specific miRNAFor further analysis of the five insects miRNA gene family,we obtained the miRNA sequences of 25 species based on the evolutionary relationship,including five vertebrate species(Homo sapiens,Mus musculus,Gallus gallus,Xenopus tropicalis,Danio Rerio),17 insects(Apis mellifera,Nasonia vitripennis,Drosophila melanogaster,Anopheles gambiae,Culex quinquefasciatus,Aedes aegypti,Heliconius melpomene,Manduca sexta,Bombyx mori,Tribolium castaneum,Acyrthosiphon pisum,Chilo suppressalis,Tryporyza incertulas,Spodoptera exigua,Plutella xyllostella,Laodelphax striatellus,Nilaparvata lugens),Caenorhabditis elegans,Daphnia pulex and Capitella teleta.As a result,14 miRNA families were found to be conserved in all species,and 9 miRNA families were found to be conserved in invertebrate species.We found four insect-specific miRNA families including mir-14,mir-277,mir-316,and mir-932.These four miRNA families are highly conserved in all insects.3.Regulation of insect-specific miR-14 in the ecdysone biosynthesis pathwaymiR-14 was selected for the functional analysis.Twelve insects with miRNA genes in miRBase were used for this analysis.The 3'UTR data for these 12 species were downloaded from the UTRdb.The target prediction software,miRanda and RNAhybrid, were used.The prediction result showed that most of genes in the ecdysone synthesis pathway were targeted by miR-14.For further analysis,we constructed insect ecdysone upstream regulatory pathways,biosynthesis and downstream response pathway(hereinafter referred to as insect ecdysone pathway)according to the published references.Twelve genes in silkworm ecdysone pathway were predicted to be target of miR-14.To validate the relationship between miR-14 and target gene in silkworm,the 3'UTR of target genes were constructed to the pMIR-REPORT plasmid,then co-transfected the HEK293T cell with miR-14 and NC mimics.The mutation plasmids were also constructed. The results showed that miR-14 inhibited the expression of InR,Akt,Erk,Torso,Nvd,Shd, EcR,E74 and Brc.InR,Akt,Torso and Erk located in the three insect ecdysone upstream regulatory pathway,Nvd and Shd located in the ecdysone biosynthesis pathway,EcR,E74 and Brc located in the downstream response pathway.All results indicated that miR-14 controlled the ecdysone biosynthesis by regulating the expression of genes in the ecdysone pathway.4.Expression patterns of miR-14 and target genesExpression pattern of miR-14 were analyzed in the head,prothoracic gland,midgut,malpighian tube and epidermis of silkworm.The abundance of miR-14-5p and miR-14-3p showed the similar trend in all the tissues,suggesting that miR-14 was a constitutive expressed miRNA.Ecdysone was only synthesized in the prothoracic gland.But miR-14 expressed highly in other tissues besides prothoracic gland,indicating that miR-14 may also have other functions.Developmental expression profile of miR-14 were also analyzed.The samples were selected every 6 h from larvae at the first day of 3rd instar to the first day of 4th instar.The result indicated that the expression profile of miR-14-3p and miR-14-5p were different from larvae to pupa,which showed a regular change within every instar conversion process.miR-14-3p increased in the first day of every instar,miR-14-5p showed an increasing profile 24h before ecdysis,and achieved the peak 12h before ecdysis.Expression profile of seven target genes were analyzed from the first day of the 3rd instar to the second of pupa,InR and Ras did not show significant variation,E74 increased in the last day of each instar,Brc increased in the pupa.The abundance of Spo,Shd,EcR showed the similar tendency from larvae to pupa,increasing from the last second day before ecdysis.Spo achieved the peak 30 h before ecdysis.Combining the expression profile of miR-14 and the target genes,we found that miR-14-5p was expressed negatively correlated with Spo,Shd,and EcR.Considering the same trends for expression profile of EcR,Spo,Shd and ecdysone titer,we concluded that miR-14-5p regulate the ecdysone biosynthesis by targeting the ecdysone pathway genes.5.Over-expression and knockdown of miR-14 affect the silkworm metamorphosisAgomir are synthetic RNA duplexes which are used for miRNA mimics.The agomir-14 was injected to the second day of 3rd instar larvae.According to the qRT-PCR result,24h after the injection,the expression level of miR-14-5p increased 3,8 and 22 times in the three biological duplicates,respectively.Different concentration of agomir-14 injection resulted in delayed ecdysis in the silkworm.About 10%of the silkworms was delayed in molt after injecting the 540 pmol agomir-14-5p,and 60%were delayed in molt for 1080 pmol agomir-14-5p.But for the agomir-14-3p group,it was the same as the control.All results indicated that miR-14-5p played the major role in the silkworm.Antagomirs are used to silence miRNA.The antagomir-14 were synthesized and then injected to the first day of 3rd instar larvae.The expression level of miR-14-5p on the 24h and 48h after injection were detected.The result indicated that the abundance of miR-14-5p was suppressed,especially at the 48h.The injection of antagomir-14-5p induced 20-30%of the silkworms with delayed molting,but the antagomir-14-3p group was similar to the control.Based on these findings,we proposed that miR-14-5p participates in silkworm metamorphosis development through the suppression of ecdysone pathway.