Effect Of Differentially Expressed MicroRNAs On The Development Of Helicoverpa Armigera And Microplitis Mediator

The cotton bollworm,Helicoverpa armigera(Hubner)(Lepidoptera:Noctuidae),is one of the most serious polyphagous insect pests of numerous crop plants and has a widespread distribution around the world.As a solitary larval endoparasitoid,Microplitis mediator(Haliday)(Hymenoptera:Braconidae)usually plays a significant role in the biological control of H.armigera in the field.MicroRNAs(miRNAs)are single-stranded,small(about 18-24 nucleotides)RNAs,they control the expression of target genes through binding to complementary target "seed match" sites within the 3' or 5' untranslated region(UTR)of mRNA targets.These miRNAs play some crucial roles in regulating at the post-transcriptional gene expression level lead to mRNA degradation,translational repression and even upregulation.Our studies focus on the miRNAs in H.armigera after parasitized by M.mediator through high-throughput sequencing,and the functions of differential miRNAs.We wanted to provide theoretical basis to reveal the opportunity to use miRNA in genetically modified plants and potentially provides new targets for biological control method of using insect miRNAs along with the parasitoid.There were the following sections in this doctoral dissertation:(1)Firstly,we selected the non-parasitized and parasitized H.armigera,constructed two miRNA libraries of H.armigera by using Illumina Solexa sequencing technology.The tested results showed that,the length of miRNAs were about 18-26 nucleotides in the two miRNA libraries.At the P<0.05 level,there were 34 notable increased and 21 notable decreased differential miRNAs.(2)Quantitative real-time PCR(qRT-PCR)is being widely used in miRNA expression studies.Suitable reference genes are necessary for the correct analysis of results.In this study,based on the sequencing results,ten candidate genes of H.armigera were selected and analyzed for their expression stability under different biotic and abiotic conditions with three statistical methods,including geNorm,NormFinder and Bestkeeper.Combination the best number of reference genes was calculated by geNorm.One target gene,let-7,was used to validate the selection of reference genes.The suitable candidate reference genes were shown as follows:miR-9 and U6 snRNA for developmental stages,miR-100 and U6 snRNA for larval tissues,miR-100 and miR-305 for adult tissues,miR-9 and miR-279 for parasitic treatment,miR-998 and U6 snRNA for nuclear polyhedrosis virus(NPV)infection,miR-9 and U6 snRNA for insecticide treatment,miR-92a,miR-100 and miR-279 for temperature treatment,miR-92a,miR-305 and miR-998 for starvation treatment,miR-9 and miR-279 for light treatment,miR-305 and miR-998 for hormone treatment.There wasn't one reference gene suitable for all samples.(3)In this study,we identified a miR-133,which was differentially expressed between non-parasitized and parasitized H.armigera.The main results showed that the expression of miR-133 was up-regulated after parasitization,quantitative real-time PCR,along with a dual luciferase reporter assay and the rescue experiment,demonstrated that Death-associated protein kinase 1(DAPK1)was the target gene of miR-133.Up-regulating the expression of miR-133 or knockdown the transcription of DAPK1 led to a significant reduction in larval growth of non-parasitized H.armigera just like the body change phenotype after parasitization,while suppressing the expression of miR-133 increased the body weight of the non-parasitized larvae.These results indicated that miR-133 involved in the body weight change of H.armigera through DAPK1.The two important immune-related genes,Relish and Dorsal,were up-regulated in the non-parasitized H.armigera after the injection of miR-133 agomir or antagomir,while they were down-regulated in the parasitized H.armigera.(4)Let-7 plays a crucial role during the development from one stage to the next,whcih affects the growth and metamorphosis of insects.In this study,we up-or down-regulated let-7 in H.armigera parasitized by M.mediator or not by using let-7 agomir/antagomir.The main results showed that the expression of let-7 was down-regulated after parasitization,deficiency or overexpression of let-7 led the pupa percentage of H.armigera reduced significantly and the parasitoids failed to make cocoons from the host body.Quantitative real-time PCR,along with a dual luciferase reporter assay,demonstrated that Broad isoform Z7(BRZ7)was the target gene of let-7.Testing three important genes from 20E and JH,earlier response gene(E74),ultraspiracle protein(USP)and intracellular receptor methoprene-tolerant(Met)showed that,both USP and Met expressions were directly regulated by let-7 in non-parasitized H.armigera,E74 expression was directly regulated by let-7 in parasitized H.armigera.We speculate that let-7 is regulated by BRZ7 through 20E and JH signaling.(5)MiR-1175 was significantly up-regulated after the parasitizing,quantitative real-time PCR and dual luciferase reporter assays confirmed the target gene of miR-1175 was Insulin-like growth factor receptor type 1(IGFIR)in H.armigera.Up-or down-regulation of miR-1175 did not affect the development of non-parasitized H.armigera or the parasitoid larvae in the host.Our study also speculated that miR-1175 can be involved in regulation the expression of Insulin receptor(InR),ProteinKinase B(Akt)and forkhead box O transcription factor(FOXO)in insulin signaling through IGF1R.