| Parasitoid wasps are parasitic insects in nature that parasitize many insects,they have been therefore widely used as natural enemies in biological control of agricultural and forestry pests.The endoparasitoid wasp,Microplitis mediator(Hymenoptera: Braconidae),parasitizes a lot of pests.M.mediator delivers a variety of parasitic factors,such as venom and PDV,which regulate immunity and development of host insects.As an important parasitic factor,the components and functions of venom of M.mediator are not clear.In this study,the components of venom proteins from M.mediator were identified by transcriptome and proteome analysis.The differentially expressed proteins of hemolymph from parasitized and non-parasitized Helicoverpa armigera were analyzed by i TRAQ-based quantitative proteomic technique.The function of a small G protein Mm_Rho1 that is present in the hemolymph of parasitized H.armigera was studied.The results are as follows:1.Transcriptome and proteome analysis of M.mediatorThe c DNA libraries of M.mediator were constructed,sequenced and analyzed.We totally acquired 18,883 unigenes and 10,731 unigenes were annotated.Based on analysis of transcriptome and proteome,2264 and 321 proteins were identified in the samples of venom apparatus(VA)and venom reservoir(VR),respectively.We found these proteins have high similarity with venom proteins from other parasitoid wasps.These venom proteins fall into four categories: enzymes,enzyme inhibitors,paralysis factors and others.1077 transcripts of venom apparatus proteins(2264)are highly expressed in the venom apparatus.A total of 75 proteins are identified in the venom.They are highly expressed in venom apparatus and have signal peptides for secretion.521 differentially expressed proteins were found in the hemolymph from parasitized and non-parasitized H.armigera by i TRAQ-based quantitative proteomic analysis.Among them,10 proteins were identified from M.mediator,including calreticulin,small G protein,metalloproteinase,etc.From 511 differentially expressed proteins,346 proteins were upregulated in hemolymph from parasitized H.armigera and 165 proteins were highly expressed in the hemolymph of the non-parasitized.KOBAS(KEGG orthology based annotation system)analysis revealed that these differential proteins are involved in physiological pathways,including immune response,material metabolism and extracellular matrix receptor interaction.Many of the key enzymes of glucose metabolism and lipid metabolism were significantly upregulated after parasitism,such as pyruvate kinase,enoyl-Co A hydratase.In summary,we analyzed and identified the venom proteins of M.mediator.The proteins in the hemolymph of H.armigera significantly changed after parasitism.2.Functional study of Mm_Rho1A small G protein of M.mediator,named Mm_Rho1,was found in the hemolymph from parasitized H.armigera by i TRAQ-based quantitative proteomic analysis.Quantitative PCR result showed Mm_Rho1 is highly expressed in the venom glands and ovaries of the wasp.The percentage of cocoon formation was reduced after RNA interference of Mm_Rho1.To investigate the function of the Mm_Rho1,recombinant protein was expressed in E.coli and purified.First,we found that number of mature eggs and total number of eggs laid in the host significantly reduced after RNA interference.The expression levels of vitellogenin was also considerably decreased after RNA interference.Expression of Vitellogenin was regulated by hormones.After RNA interference,the titer of ecdysone increased.The expression of juvenile hormone synthesis related genes was down-regulated,but the expression of juvenile hormone metabolism related genes had no change.Accordingly,the titer of juvenile hormone decreased.Secondly,we asked whether Mm_Rho1 affects the host’s cellular immune responses.The results showed that the spreading,phagocytosis and encapsulation of host’s hemocytes were suppressed by the recombinant Mm_Rho1 protein.We further proved that Mm_Rho1 interacted with H.armigera Dia,a cytoskeleton-related protein,by yeast two hybrid assay and pull-down assays.In summary,we found that Mm_Rho1 regulates the titer of the hormones of parasitoid wasp and the development of their eggs.In addition,Mm_Rho1 regulates cellular immunity through interaction with the Dia protein in the host. |
PDF Full Text Request