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Identification And Function Analysis Of Olfactory Proteins In Oedaleus Asiaticus (Orthopera:Acrididae)

Posted on:2020-07-30Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y T ZhouFull Text:PDF
GTID:1363330578956526Subject:Crop Protection
Abstract/Summary:PDF Full Text Request
The band-winged grasshopper,Oedaleus asiaticus Bei-Bienko,is one of the most dominant and economically important grasshopper species in the steppe grasslands and farming-pastoral ecotone in northern China,and often requires chemical control during outbreaks.This species forages mainly on gramineaceous plants,and since it prefers to overgrazed steppes and xerophytous habitats,it has been suggested as an indicator species for steppe deterioration in Inner Mongolia.Insects identify various odor molecules from the environment by olfactory sensory system,so as to guide insects to forage,mate,locate oviposition sites,avoid predators and other life activities.Insects detect semiochemicals through interactions between various olfactory proteins,so olfactory proteins are the molecular basis of olfactory activity in insects.However,up to now,there is little knowledge about the olfactory function in this grasshopper.Therefore,the identification and functional study of olfactory protein genes are conducive to clarify its chemosensory mechanism,and provide the theoretical basis for finding the molecular targets for green control of locusts.In this study,we used the antennal transcriptomes data of O.asiaticus to identify olfactory protein genes.Then,we conducted a further analysis of bio informatics and expression profiles.After prokaryotic expression of major chemosensory proteins was performed,the ligand binding ability of the proteins were determined.At last,RNA interference(RNAi)was combined with electroantennogram(EAG)reaction to detect interference effects.The main results are as follows:1.Identification of olfactory protein genes of O.asiaticusBased on the antennal transcriptomes data of O.asiaticus assembled in our lab,a total of 86 olfactory protein genes were identified by bioinformatics methods,including 17 CSPs,60 ORs(59 OR and ORco),6 IRs and 3 SNMPs.Bioinformatics analysis indicated that,except for OasiCSP19,the other 16 OasiCSPs have complete open reading forums(ORFs)ranging from 372 bp to 516 bp.Multiple alignment of the amino acid sequences of all the 17 OasiCSPs indicated that these OasiCSPs contain four conserved cysteine residues.Of this 60 ORs,only three putative OR genes,OasiOR14,OasiOR15 and OasiORco,had complete ORFs with lengths of 1350 bp,1194 bp and 1146 bp,respectively,and their encoded proteins contained 4-7 transmembrane domains with an inside or outside N-terminus.All of six candidate IRs had one to four transmembrane domains,and other four IRs except OasiIR1 and OasiIR76b had signal peptides with 16 to 19 amino acids.OasiIR8a and OasiIR25a had a full-length ORF.These SNMPs had one to two transmembrane domains.Multiple alignment of the amino acid sequences indicated that SNMPs contained six conserved cysteine residues.2.Expression profile analysis of olfactory protein genes of O.asiaticusqRT-PCR was performed to determine the expression levels of olfactory genes in different adult tissues(antenna,head without antennae and mouthparts,labrum,labium without labial palps,labial palp,maxillary palp,thorax,tarsus,wing and abdomen)of both males and females.The results showed that there were significant differences in the expression levels of eight different CSP genes in different adult tissues.Especially,OasiCSP8 was much highly expressed in the labial palps and maxillary palps,and OasiCSP11 and OasiCSP13 had the highest expression levels in the antennae.OasiCSPl5 had much higher expression levels in chemosensory tissues(antenna,labrum,labium without labial palps,labial palp,maxillary palp,and tarsus)than in non-chemosensory tissues(head without antenna and mouthpart,thorax,wing,and abdomen).However,OasiCSP12 had similar expression distribution in nearly all the tested tissues.14 OR and two SNMP genes(OasiSNMP1 and OasiSNMP2a)were strongly expressed in adult antennae,and nearly all tested genes displayed significant differences in the expression levels between both sexes.Moreover,two IR genes(OasiIR25a,and OasiIR76b)had uniquely high expression levels in the antennae,labial palps and maxillary palps,while three IR genes(OasilR1,OasiIR2 and OasiIR3)were highly expressed in most tested tissues(head without antennae and mouthparts,labial palp,maxillary palp,labium without labial palps,thorax,tarsus,and abdomen),but OasiIR25a was just faintly expressed in the antennae,labia without labial palps,labial palps,maxillary palps and abdomens.3.Fluorescence competitive binding assays of CSPs of O.asiaticusThe recombinant expression plasmid for 8 OasiCSPs was constructed and successfully expressed in Escherichia coli,and the proteins were purified using a Ni column.The bbinding affinity of these eight recombinant CSP proteins was measured for 16 volatiles from the host plant(Stipa krylovii),fecal and live body of adult O.asiaticus by the fluorescence competitive binding assays.The results showed that 8 CSP proteins displayed different degrees of binding affinities to various volatiles.Except for OasiCSP11 displaying the highest binding affinity to one live body volatile(2-ethylhexanol),with Ki value of 9.84 ?M,OasiCSP12 showed higher binding affinities for most volatiles than other 7 OasiCSPs.OasiCSP12 exhibited strong binding affinities for five host plant volatiles(hexanal,(Z)-3-hexen-1-ol,butylacetate,ethylbenzene,mesitylene)and one volatile(2-ethylhexanol)from the live body of adult O.asiaticus(Ki<20 ?M),especially butylacetate and(Z)-3-hexen-1-ol,with the Ki values of 13.73 and 14.73 ?M,respectively.OasiCSP 15 showed the weakest binding affinities for the tested compounds and only combined two compounds.4.RNA interference(RNAi)effects on chemosensory proteins of O.asiaticusTo further verify their olfactory functions,RNA interference(RNAi)and electrophysiological recording were conducted.Three dsCSPs(dsCSP4,dsCSP11,and dsCSP12)were injected into the male and female adults of O.asiaticus,respectively.After 48 h,qRT-PCR results showed that the injection of dsCSP12 significantly reduced the expression levels of OasiCSP12 in both male and female antennae,compared to water-injection and non-injection control groups.After injection of dsCSP4 and dsCSP11,the expression levels of OasiCSP4 and OasiCSP11 were significantly decreased in females,but not significantly in males.Sixteen compounds were used to detect the EAG reaction of male and female adults after different injection treatments showed that after injecting dsCSP4,for females,the EAG responses showed significant reductions in response to three compounds((E)-2-hexenal,(Z)-3-hexen-1-ol and methacrolein).For males,the dsCSP4-injected antennae exhibited significant decrease in response to 12 compounds.It was also found that 2-methylbutyraldehyde could evoke stronger responses after RNAi.Silencing OasiCSP11 significantly decreased the EAG responses in females to 13 compounds,while male antennae only significantly reduced the EAG responses to 4 compounds.Injection of dsCSP12 in females and males caused the most obvious EAG responses to all odorants.The EAG responses of all 16 compounds should be remarkable ly reduced in female antennae.In addition to(E)-2-hexenal,(Z)-3-hexen-1-ol and heptaldehyde,the EAG reaction of male antennae for other 13 compounds was also significantly reduced.These results suggest that OasiCSP4,OasiCSP1 and OasiCSP12 may play important roles in finding host plants and aggregation in this species.
Keywords/Search Tags:Oedaleus asiaticus, Olfactory protein, Expression profile, Fluorescence competitive binding, RNA interference, EAG response
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