Analysis Of Venom Proteomics And Four Venom Protein Physiological Functions In Pteromalus Puparum

Parasitoids are natural enemies of numerous insects that as important biological control agent to achieve pest status in agriculture system. Its deposit their eggs within the host insect along with virulence parasitic factors such as venom, polydnaviruse (PDV), virus-like particle (VLP), and teratocyte to interfere with host’s immune response or to disrupt host’s development to ensure their offspring successful development of in the hemocoel. The parasitic factors such as venom would be potentially valuable resources on developing new environmentally safe insect immune-suppression agents or transgenetic crops with modern biotechnologies. So, searching the parasitoid virulence parasitic factors and determining the functions of parasitic factors would help the next development. Up to date, only some parasitoids venom protein has been documented and only little venom protein has been studied. In this study, we chose Pteromalus puparum (Hymenoptera:Pteromalidae), a predominate pupal endoparasitoid of Pieris rapae (Lepidoptera:Pieridae) as object, combined venom gland transcriptome and venom proteomic method to analysis the constituents of Pteromalus puparum venom. And we selected some venom protein to expression in bacterial, and prepared rabbit polyclonal antibodies, studied their expression pattern and verified their biological function on its host.1. The constituents of Pteromalus puparum venomWe sequenced the transcriptome of P. puparum venom gland and other tissue firstly, and analysed the transcriptome data. Then we used shotgun proteomic method to analysis P. puparum venom protein, the obtained data blasted with the database of P. puparum transcriptome and Metazoa database. And we obtained60venom protein from P. puparum venom, divided them into5types, including enzymes, proteinase inhibitors, recognition or binding proteins, unknown proteins and other.2. Functional analysis of calreticulin from Pteromalus puparum venom and its relationship with calreticulin of Pieris rapaeWe found a Coiled-coil domain in C terminal, and expressed the wild-type and the Coiled-coil domain deleted mutant P. puparum CRT (PpCRT) in bacterial and prepared the polyclonal antibody against PpCRT. RT-PCR results showed PpCRT gene expressed highest in venom gland, and the highest expression level in venom gland at after emergency2days. Western blotting analyses showed that PpCRT not only existed in venom but also in all tissues, and PpCRT lasted in venom after emergency. Recombinant His-tag wild-type and mutant PpCRT could unequally bind to P. puparum egg surface. PpCRT entered Pieris rapae hemocytes, and the Coiled-coil domain was important for entering hemocytes. Recombinant PpCRT inhibited host P. rapae hemocyte spreading and cellular encapsulation in vitro, and the Coiled-coil domain of PpCRT affected PpCRT exerting function. Injection PpCRT into P. rapae pupa, real time PCR results showed PpCRT inhibited P. rapae encapsulation-related genes (CRT, scavenger receptor) expression. PrCRT mRNA in hemocytes was significantly induced after injection of yeast or beads, but did not change noticeably after injection of Escherichia coli or Micrococcus lysodeikticus. Recombinant PrCRT enhanced cellular encapsulation by P. rapae hemocytes in vitro, and the N-domain of PrCRT was required for encapsulation. RNAi of PrCRT by dsRNA injection impaired the ability of hemocytes to encapsulate beads. After parasitization by P. puparum, PrCRT mRNA and protein levels in P. rapae pupal hemocytes were significantly suppressed compared to non-parasitized control.3. Isolation and functional analysis of Pacifastin type serine protease inhibitor from Pteromalus puparum venomWe isolated the Pteromalus puparum venom by RP-HPLC, and obtained a single venom component. After MS/MS and N sequence, we identified the No.2peak as P. puparum venom Pacifastin type serine protease inhibitor (PpPI). We cloned the full sequence of PpPI, expressed in bacterial and purified, prepared the rabbit polyclonal antibody. RT-PCR results indicated PpPI gene special expression in venom gland and the highest expression level in venom gland at after emergency2days. Western blotting results showed PpPI special existed in venom protein and the highest level in venom at after emergency3days. We tested the recombinant or chemical synthesis PpPI effect on host or non-host hemolymph PO activity or PPO activation. Results showed PpPI had no effect on hemolymph PO activity, and inhibited the hemolymph PPO activation of host Pieris rapae and Papilio xuthus. PpPI also had no effect on hemolymph PO activity, inhibited the hemolymph PPO activation of non-host Bombyx mori. PpPI had no effect on hemolymph PO activity and PPO activation of non-host Chilo supperssalis. Interesting, PpPI enhanced the hemolymph PPO activation of non-host Manduca sexta. Through serine proteinase assay, PpPI inhibited trypsin activity, but had no effect on chymotrypsin and ptoteinase K activity.4. Functional analysis of Kazal type serine proteinase inhibitor from Pteromalus puparum venomWe cloned the full sequence of P. puparum venom Kazal type serine proteinase inhibitor (PpKazal), expressed in bacterial and purified, prepared the rabbit polyclonal antibody. RT-PCR results showed PpKazal gene special expression in venom gland and the highest expression level in venom gland at after emergency2days. Western blotting results showed PpKazal special existed in venom protein, and lasted in venom through adult period. PpKazal had no effect on hemolymph PO activity, and inhibited the hemolymph PPO activation of host Pieris rapae and Papilio xuthus.5. Characterization of general odorant binding protein from Pteromalus puparum venomWe cloned the full sequence of P. puparum venom general odorant binding protein (PpGOBP), expressed in bacterial and prepared the rabbit polyclonal antibody. PpGOBP gene special expression in venom gland and the highest expression level in venom gland at after emergency2days. PpGOBP existed in venom gland and venom, and its expression stable in venom at adult stage. Compared with non-mated, PpGOBP gene expression in venom gland after mated had no significant difference. After feeding honey or parasitized, PpGOBP gene expression in venom gland was siginificant promoted. Using PpGOBP polyclonal antibody, we detected PpGOBP located in venom gland.Combined transcriptome and ptoteomic methods, we idenfified the constituents of Pteromalus puparum venom and obtained60venom proteins. We selected calreticulin, Pacifastin type serine protease inhibitor, Kazal type serine proteinase inhibitor and general odorant binding protein to study their expression pattern and biological functions. This study made a great contribution to gain insights into understanding parasitoid venom components and made the foundation for exploiting venom protein for agricultal insect control.