| The small cabbage butterfly. Pieris rapae, is an important pest of cruciferous corps, and Pteromalus puparum is a predominant pupal endoparasitoid wasp of this butterfly (Hu,1983). For successful development of parasitoid offspring, female parasitoids usually introduce one or several kinds of maternal factors into the hemocoels during oviposition to suppress host immunity (Pennacchio et al.,2006). In endoparasitoids without PDVs, venom serves as the key suppressor of host immunity (Asgari et al..2011). Aside from suppressing immune reactions, parasitoids also re-direct host developmental processes via influence on genes encoding many neuropeptides (Shi et al.,2015). We reported that P. rapae immune responses, determined by encapsulation of Sephadex beads, was reduced as early as30min following artificial envenomation by injecting P. puparum venom into experimental pupae (Fang et al.,2010). This study is the first exploration to characterize transcriptome profiles of P. rapae pupae using high-throughput illumina RNA sequencing, and investigate earlyier stage changes in the host transciptome after parasitization by endoparasitoid, P. puparum. And we identified and characterized three venom proteins from P. puparum venom gland transcriptome, this study provide the foundation for further investigation on the physiological function of these three venom proteins.1. To investigate the early changes in host immune-related genes following parasitization, we analyzed transcriptomes of parasitized and unparasitized, control, host pupae. Approximately17.7million and19.3million paired-end reads were generated from non-parasitized and parasitized host pupae, and assembled de novo into45,639transcripts and27,659non-redundant unigenes. The average unigene length was790bp. A total18,377of27,659unigenes were annonated, and557differentially was identified expressed unigenes in host pupae at1h after parasitization, of which21were immune-related. Parasitization led to down-regulation of most pattern recognition receptors and up-regulation of all serine protease inhibitors. The transcirptomic profile of P. rapae is considerably affected by parasitization. This study provides valuable sources for future investigations of the molecular interaction between P. puparum and its host P. rapae.2. Gamma-glutamyl tanspeptidase (GGT) was ubiquitous in oganisms. These enzyme involved in glutathione metabolism and play imptant role in antioxidant defence, detoxification and inflammation processes. We analyzed a P. puparum venom gland proteome and transcriptome and identified a partial gene encoding a possible Gamma-glutamyl tanspeptidase. According to the comparatively transcriptomic data between the venom gland and body carcass, the transcripts levels of PpGGT genes were significantly high in venom gland. The full length PpGGT sequences were2274bp and encoded652aa. The predicted results showed that theoretical isoelectric points of PpGGT was8.25, and the related molecular weights was72.196kDa. Based on the multiple sequences alignment results, PpGGT precursor can autoproteolytic into a small and large subunit. Real time quantitative RT-PCR. western blot and immunohistochemistry results verified that the mRNA expression levels of the PpGGT genes were remarkable high in venom gland, lmmunoblotting results indicated that the contents of PpGGT could only be detected in venom protein mixture. A time-course analysis showed that the expression of PpGGT peaked at day6post-eclosion. GGT activity can be detect in venom and in the recombinant PpCBP, we suggest that this venom protein may induce host hemocyte apoptosis by affect the GSH metabolism.3. Chitin-binding proteins (CBPs) are present in many species and they act in a variety of biological processes. We analyzed a P. puparum venom gland proteome and transcriptome and identified a partial gene encoding a possible chitin-binding protein. Here, we report cloning a full length cDNA of a sequence encoding a chitin-binding-like protein (PpCBP) from P. puparum. The cDNA encoded a96amino acid protein, including a secretory signal peptide and a chitin binding peritrophin-A domain. Phylogenetic analysis of chitin binding domains (CBDs) of cuticle proteins and peritrophic matrix proteins in selected insects revealed that the CBD of PpCBP clustered with CBD of Nasonia vitripennis CBP and of Fenneropenaeus merguiensis shrimp ovarian peritrophin. The PpCBP is specifically expressed in venom apparatus of P. puparum. A time-course analysis showed that the expression of PpCBP peaked at day4post-eclosion. Binding assays showed the recombinant PpCBP selectively binds chitin, but not cellulose in vitro. We infer that PpCBP serves as a structural component of the venom reservoir to protect it from the bioactive proteins produced by venom gland.4. Serine proteinaes homologs (SPHs) play key roles in many important physiological processes of insect. We analyzed the P. puparum venom gland proteome and transcriptome and identified a partial gene encoding a possible PpSPH29. In this study, the full length cDNAs of PpSPH29were cloned from the mRNA isolated from the venom gland of a pupal endoparasitoid, P. puparum. According to the comparatively transcriptomic data between the venom gland and body carcass, the transcripts levels of PpSPH29genes were significantly high in venom gland. The full length PpSPH29sequences were1065bp and encoded270aa. The predicted results showed that theoretical isoelectric points of PpSPH29was4.4, and the related molecular weights was29.4kDa. Based on the multiple sequences alignment results, PpSPH29possessed a catalytic domain or its isoform at the C-terminus. It was interesting that for PpSPH29the third catalytic triad residue "Ser" were replaced by "Gly". Real time quantitative RT-PCR results verified that the mRNA expression levels of the PpSPH29genes were remarkable high in venom gland. Immunoblotting results indicated that the contents of PpSPH29could only be detected in venom protein mixture. The outcomes acquired from our research are the preparation for the functional studies of SPHs from parasitoid venom and will give a further insight into the comprehension of SPHs in insects. |
PDF Full Text Request