Identification Of Venom Proteins From Nasonia Vitripennis And Functional Analysis Of PACIFASTIN And KAZAL Type Genes

Flies are varied and widely distributed, and is one of the most important sanitary pest in the world. They can carry a variety of pathogens and spread many diseases between human and livestocks. Especially in the residential area, Musca domestica, accounted for nearly ninety percent, seriously affect the public health and harass human life. Nasonia vitripennis is the ectoparasitoid wasp of higher diptera flies pupae. The female wasps inject venom into host when make the oviposition to make sure the successful growth of their offsprings.’Their venom can regulate and inhibit the growth and immune response of host effectively. Nasonia vitripennis, with less chromosome numbers and raise conveniently, is also one of the ideal model insects. The researches on its venom and interaction studies between wasps and hosts can not only provide an effective biological method for flies control, but construct an excellent model system for further study of the influence of an ectoparasitoid venom on its pupal host. In this study, we set the Nasonia vitripennis and Musca domestica as the object, combined transcriptomics, proteomics and suppression subtractive hybridization methods to analyse the venom components of Nasonia vitripennis, and unstand the venom influence on genes transcript of Musca domestica hemocytes. Meanwhile, four venom genes were seleted to make the prokaryotic expression, protein purification and functional study in vitro. The contents and results of this study are as follows,1Gene transcript profiling of Musca domestica pupae hemocytes treated by Nasonia vitripennis venom in vitroForward and reverse suppression subtraction libraries were constructed of hemocytes of Musca domestica pupae, which have been treated by venom and Sephadex A-50for1h. Ultimately,133down-regulated and111up-regulated ESTs were submitted to GenBank, accession numbers are from JZ152413to JZ152656. For obtained genes, functional annotation were marked and classificated into9categories, including immune response; cytoskeleton; cell cycle and apoptosis; respiration and energy metabolism; material metabolism; transport; stress response; and transcriptional and translational regulation as well as unknown contigs. Meanwhile, we randomly seleted8genes from forward and reverse libraries to make the confirmation by Real-time quantitative PCR respectively, and also determined the PO, Cu2+/Zn2+SOD and ATPase activities. The consistency of all results showed the credibility of our SSH library construction.2Transcriptome determination of venom gland and other residual tissues of Nasonia vitripennisTranscriptome of venom gland and other residual tissues in Nasonia vitripennis were determined after dissection by RNA-seq methord. A total of52907Unigene (51805from venom gland and52654from residual tissues) were obtained, of which38619Unigene were annotated. In addition, functional classification were made by clusters of orthologous groups(COG) and gene ontology(GO) methords.3Proteome determination of venom of Nasonia vitripennisProteome of Nasonia vitripennis were determined by LC-MS/MS method. On the one hand,22bands were cut after SDS-PAGE of total venom protein and identified by mass spectrum after enzymolysis. On the other hand, the total venom protein were taken directly to do the reversed phase high performance liquid chromatography analysis (RP-HCLP), then the unimodal constituent was identified by mass spectrum respectively. All the obtained data were blasted to the transcriptome data of Nasonia vitripennis. As a result,71effective venom components were abtained. We classified them into six types, which are Proteases and peptidases; Protease inhibitors; Recognition/binding proteins; Stress response proteins; Others and Unknown proteins.4Expression profiling and functional analysis of two PACIFASTIN type genes in Nasonia vitripennisSeveral primers were designed according to Nasonia vitripennis genome sequences to clone two Pacifastin type serine protease inhibitor genes, nvpp-land nvpp-2, which were723bp and888bp length with the whole open reading frame coding240and295amino acid sequences respectively. They both have one signal peptide sequence consisted of17amino acid residues in N terminals. Sequence analysis and comparison showed that nvpp-1and nvpp-2contained5and4typical Pacifastin conservative domains respectively. Transcriptional and expressional profiles of nvpp-1and nvpp-2in different tissues and developmental stages in its venom of Nasonia vitripennis were analysed by Real-time quantitative PCR and Western bloting mathods. The results indicated that nvpp-1and nvpp-2transcribed in each tissues of Nasonia vitripennis, especially in its thorax, residual abdomen and venom gland. In venom gland, they both highly transcribed at freshly eclosioned stage, and then appeared a strongly downward trendency. However, NVPP1and NVPP2were both expressed in venom but not in other tissues or rarely expressed. Meanwhlie, prokaryotic expression of NVPP-1and NVPP-2were operated by vector pET-28a (+), and their inhibiting effection on trypsin, chymotrypsin and proteinase K were tested by pured and refolded NVPP-1and NVPP-2proteins. In addition, the influence on PO activity and PPO activation of hemolymph of Musca domestica pupae, the host of Nasonia vitripennis, were studied by pured NVPP-1and NVPP-2proteins. The results demestrated that NVPP-1and NVPP-2can not only inhibit the activities of chymotrypsin and trypsin respectively,but supress PPO activation progress in pupae hemolymph of Musca domestica.5Expression profiling and functional analysis of two KAZAL type genes in Nasonia vitripennisSeveral primers were designed according to Nasonia vitripennis genome sequences to clone two Kazal type serine protease inhibitor genes, nvkvp-1and nvkvp-2, which were198bp and264bp length with the whole open reading frame coding65and87amino acid sequences, and have the signal peptide sequence consisted of19and21amino acid residues in N terminals respectively. Sequence analysis and comparison showed that nvkvp-1and nvkvp-2both have one typical Kazal conservative domains. Transcriptional profiles of nvkvp-1and nvkvp-2in different tissues and developmental stages in its venom of Nasonia vitripennis were analysed by Real-time quantitative PCR. The results indicated that nvkvp-1and nvkvp-2transcribed highly in its venom gland. They both transcribed little at freshly eclosioned stage, then increased in the second day and decreased in the third day, especially in the fourth day, the transcript level reached the maximum,then decreased again until the seventh day. Meanwhlie, Prokaryotic expression of NVKP-1and NVKP-2were operated by vector pGEX-4T-2, and their inhibiting effection on trypsin, chymotrypsin and proteinase K were tested by pured proteins. In addition, the influence on PO activity and PPO activation of hemolymph of Musca domestica pupae, the host of Nasonia vitripennis, were studied by pured NVKVP-1and NVKVP-2proteins. The results demestrated that they both can supress the PPO activation progress in pupae hemolymph of Musca domestica. NVKVP-1can also inhibit the activity of trypsin, while NVKVP-2had no effect on these three proteases.This work not only illuminated the venom components of Nasonia vitripennis but understand the gene regulation of Musca domestica pupae hemocytes treated by venom. This will not only provide the theoretical basis for further researches but give new thought and method to control the flies.