Studies In Identification And Tissue Expression Profile Of Meteorus Pulchricornis Chemosensory Genes And Expression In Vitro

The parasitoid wasp Meteorus pulchricornis(Hymenoptera: Braconidae)is a dominant solitary endoparasitoid of numerous lepidopteran pests,such as Spodoptera litura,Spodoptera exigua.It is the important natural agent for effective control of Lepidoptera pests.In recent years,many insect chemosensory proteins have been identified with the rapid development of sequencing technology,molecular biology and bioinformatics.and these progresses contribute to increase the understanding of the insect chemosensory mechanism.However,the reported studies mainly focused on Lepidoptera and Coleoptera species,researches exploring the parasitic insect chemosensory systems were very limited.Like most of other insects,the parasitic insects can also locate host successfully in a complicated environment by acting a series of behavioral activities,which regulated by semiochemicals principally.The present study was aimed to ecucidate the chemosensory mechanism of parasitoic wasp and it could lead to improvement the parasitizm efficiency of M.pulchricornis females and making them play a greater role in biological control practice.It could also help us to go deep into the understanding the behavioral ecology mechanism of parasitoids.The present study include:(1)Identifications of chemosensory genes,including odorant binding proteins(OBPs),chemosensory proteins(CSPs),odorant receptors(ORs),ionotropic receptors(IRs),gustatory receptors(GRs),and odor degrading enzymes,including carboxylesterase(CXEs)and glutathione S transferase(GSTs)through the next generation transcriptome sequencing.we analysed the Sequence information of these genes were also analysed and phylogenetic trees were also constructed..Real-time quantitative PCR was performed for the validation of relative expression levels of OBPs,GRs,CXEs,GSTs.(2)The putative chemosensory genes(OBPs,CSPs,ORs,IRs,GRs)were identified from M.pulchricornis ovipositor transcriptome database through Illumina sequencing,and sequence alignment and phylogenetic tree were contructed.The real-time quantitatice PCR was operated for the M.pulchricornis ovipositor tissue expression profiles.Besides,examination and photography were performed using the scanning electron microscope for ovipositor morphological structure.(3)According to open reading frame of the antenna-specific Mpul OBP9,the gene-specific primers were designed and appropriate restriction enzymes were choosed to build the recombinant plasmid pet32a(+)/Mpul OBP9.Lots of recombinant Mpul OBP9 protein was obtained from the prokaryotic expression system.Proteins were purified by means of Ni ion affinity chromatograph.The purified proteins were digested with VI enterokinase to remove the His-tag.Finally,the target proteins were purified by means of ion exchange chromatography.We present the first M.pulchricornis antennal transcriptome database.A total of 34,967 unigenes were assembled with an N50 of 2,482 bp.The identified unigenes have the most sequence similarities with the sequence in Microplitis demolitor.We identified 16 Mpul OBPs,8 Mpul CSPs,99 Mpul ORs,19 Mpul IRs,13 Mpul GRs and 1 Mpul SNMP.The results of q PCR showed 14 Mpul OBPs(Mpul OBP1 and Mpul OBP4-16)were predominantly expressed in the antenna.It indicates that the Mpul OBPs play key roles in the recognition of olfactory cues.Mpul OBP2 and Mpul OBP3 had the highest level in the leg and abdomen,respectively,which indiacted that Mpul OBPs may involve in other physiological process in M.pulchricornis.Nine Mpul GRs(Mpul GR1,Mpul GR 3,Mpul GR 5,Mpul GR 7,Mpul GR 9,Mpul GR 10,Mpul GR 11,Mpul GR 12 and Mpul GR 13)were highly expressed in antenna.We speculate Mpul GRs may play important roles in chemorecption of M.pulchricornis.Mpul GR6 was mainly expressed in abdomen,which suggested they are likely to have potential non-olfactory functions.In order to obtain the comprehensive understanding of sensory process in M.pulchricornis,odor degrading enzymes genes(ODEs)were identified and tissue profile analysis were conducted.Totally,20 Mpul CXEs and 13 Mpul GSTs have been identified.The results of q PCR suggested that 9 Mpul CXEs(Mpul CXE1-3,Mpul CXE9,Mpul CXE 13,Mpul CXE 14,Mpul CXE 16,Mpul CXE18 and Mpul CXE20)and 4 Mpul GSTs(Mpul GST2 and Mpul GST6-8)were abundantly expressed in the antenna.By contrast,9 Mpul CXEs(Mpul CXE5,Mpul CXE7,Mpul CXE8,Mpul CXE10-12,Mpul CXE15,Mpul CXE17 and Mpul CXE19)and 9 Mpul GSTs(Mpul GST1,Mpul GST3-5,Mpul GST9 and Mpul GST10-13)had the highest expression level in the abdomen.Mpul CXE4 was mainly expression in the ovipositor.Mpul CXE6 and Mpul GST11 had similar expression patterns in the antenna and abdomen which indicated that Mpul CXEs and Mpul GSTs may be also involved in the chemosensory process and xenobiotic metabolism in M.pulchricornis.In order to explore the function of ovipositor of parasitoids in the chemosensory process.we observed the sensilla types and structure features of ovipositor using the scanning electron microscope firstly.The result indicated that there were two types of sensillum,trichoid sensillum and ctenidia sensillum.The barbs and/or serrations were also detected on the top of ovipositor valvula.Then,we constructed the M.pulchricornis ovipositor transcriptome database.There were 51,593 unigenes assembled with an N50 of 1,727,which shared high similarities with the sequence from M.demolitor.According to the bioinformatics analysis,we obtained 12 Mpul OBPs,8 Mpul CSPs,25 Mpul ORs,21 Mpul IRs,5 Mpul GRsand 5 Mpul SNMPs.The phylogenetic analysis revealed that the chemosensory gene sequence had high homology.The q PCR results showed 10 Mpul OBPs(Mpul OBP17-22,Mpul OBP24,Mpul OBP25,Mpul OBP27 and Mpul OBP28)were primarily expressed in the antenna.Mpul OBP26 was mainly expressed in the abdomen.Mpul OBP23 had similar expression between antenna and thorax.The results suggested that antenna-specific Mpul OBPs had a negative feedback regulation in the ovipositor chemoreception.The regulation of ovipositor needs to be further investigated.In view of exclusive antenna-specific expression of Mpul OBP9,we obtained the target protein by molecular cloning,prokaryotic expression and protein purification.The corrected Mpul OBP9 was sub-cloned into the bacterial expression vector pet32a(+),then expressed in E.coli BL21 induced by IPTG.SDS-PAGE analysis showed that there was obvious bold band in the supernatant of induced Mpul OBP9 / pet32a(+).Recombinant protein was purified by Ni ion affnity chromatography and enterokinase digestion with His-tag removed,which was purified by ion exchage chromatography to obtain target protein.The study will provide important basis for subsequent function research of M.pulchricornis OBPs.