Identification Of MiRNAs In Pteromalus Puparum And Analyses Of Its Ovary Development Related MiRNAs

Parasitoid wasps,an extraordinarily diverse group with broad host range,are important natural enemies of pests.Pteromalus puparum,a pupal parasitoid wasp of some butterflies including the cabbage white butterfly Pieris rapae,has a tremendous impact on biological control of insect pests in farmlands.In this thesis,we investigated miRNA biogenesis pathway genes and miRNA genes in the genome of Pteromalus puparum and analyzed the miRNA genes which were responsive to Ecdysone receptor(EcR)and Methoprene-tolerant(Met)in fat body and ovary.The studies provide theoretical basia for coordinate regulation on ovary development by hormones and miRNAs.The main results are described as follows:1.Annotation of biogenesis pathways for miRNA and siRNA in Pteromalus puparumCombined genome and transcriptome,we annotatd genes including Drosha,Pasha,Exportin-5,Dicer-1,Loquacious and Argonaute-1 for miRNA biogenesis pathway,and Dicer-2,R2D2,Argonaute-2,Sid1 for siRNA pathway.All core genes of the two pathways are present in the genome as a single copy,except for two genes in the siRNA pathway,R2D2(two copies)and Argonaute-2(three).Phylogenetic analyses of hymenopteran Dicer and Argonaute genes suggested significantly accelerated molecular evolution rates of the genes involved in the siRNA pathway.Expression analyses revealed higher mRNA levels for all miRNA pathway genes in P.puparum adults and the siRNA related genes were expressed in different patterns.2.Analysis of P.puparum miRNA genesBy constructing eight small RNA libraries from different developmental stages and genders,we identified 254 mature miRNAs originated from 75 known and 119 novel miRNA genes in P.puparum,88 of which reside in 26 clusters.Comparing miRNA expression in the eight libraries,we found 84 mature miRNAs were differentially expressed between embryos and larvae,20 between larvae and pupae,and 26 between pupae and adults.We found some miRNAs were differentially expressed between sexes in embryos(10),larvae(29),pupae(8)and adults(14).Besides,miRNA gene gains and losses were analyzed among 15 hymenopterans,results indicated losses of miR-87,miR-306 in chalcidoidea,and loss of miR-316 occurred in chalcidoidea and braconid wasps.3.miRNAs in response to RNA interference of PpEcR and PpMet in ovary and fat bodyRNA interferences of Argonaute-1(PpAgo1),PpEcR and PpMet led to deccelarated accumulation of vitellin in P.puparum ovary.qPCR results indicated the expressions of Vitellogenin-1(PpVg1)and Vitellogenin-2(PpVg2)in fat body were moderately modulated by PpEcR and PpMet separately,and the trends changed along with the development.The expression of Vitellogenin receptor(PpVgR)in ovary was not affected by PpEcR and PpMet.By interrogating miRNA levels in black pupae,24 hours adults post eclosion and 48 hours adults post eclosion,we identified 54,43 miRNAs in ovary and fat doby separately,which significantly changed in response to RNA interference of PpEcR and PpMet.In ovary,miRNAs from let-7-C and miR-3791-318 cluster were up and down regulated respectively after RNA interferences of PpMet.miRNAs from miR-2-71 cluster were up regulated after RNA interferences of PpEcR.In fat body,miR-275-305 and miR-2-71 cluster miRNAs were up regulated by RNA interference of PpMet.In contrast,the expressions of miR-2-71 cluster miRNAs were down regulated by RNA interference of PpEcR.4.Analysis of P.puparum miR-3791-318 gene clusterP.puparum miR-3791-318 gene cluster contains five miRNA genes including miR-3791,miR-279c,miR-9b,miR-2944 and miR-318.Multiple sequence alignment of miR-3791-318 clusters revealed higer conservations at the 3p arms of each miRNA genes.Expression analyses revealed higher miRNA levels in ovaries and the miRNA levels after eclosion increased when emergence and peak at 48 hours post eclosion RNA interferences of PpMet led to deccelarated miRNA levels in ovary.Three conserved E-box motif which were candidate PpMet bingding site,were found near the transcription start site(TSS).Besides,five potential E74 bingding sited were predicter near TSS.RNA interferences of PpE74A but not PpE74B led to deccelarated miRNA levels in ovary,suggesting a coordinate regulation by ecdysone.Target predictions for the mature miRNA from 3p arm were conducted and enrichment was oberserved in the Wnt pathway.5 genes from Wnt pathway were predicted as the target of miR-3791-3p,and the mRNA levels of CtBP significantly reduced after injection with miR-3791-3p mimics.The results indicated miR-3791-3p may target Wnt pathway genes and function in ovary development5.Analyses of potential miRNA interacted with PpMet Twelve miRNAs were predicted to by miRanda,TargetScan and PITA:miR-2c-5p,miR-14-3p,miR-92a-3p,miR-252-5p,miR-275-3p,miR-279b-3p,miR-307-3p,miR-927b-3p,miR-996-5p,miR-3477-3p,miR-6001-3p and let-7-3p.qPCR results revealed unchanged mRNA levels of PpMet after injection with miRNA mimics and no ovarian defects were oberserved.Besides,dual luciferase reporter assay suggested no interaction between 3'UTR of PpMet with 12 miRNAs.