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Functional Study On Close Range Recognition Of The Chemosensory Related Proteins In Legs Of The Apolygus Lucorum(Hemiptera: Miridae)

Posted on:2021-02-18Degree:MasterType:Thesis
Country:ChinaCandidate:Z B LiFull Text:PDF
GTID:2393330602990435Subject:Agricultural Entomology and Pest Control
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The Apolygus lucorum(Hemiptera:Miridae),a kind of important field pest,became major pests in cotton fields along with the widespread cultivation of Bt transgenic cotton,causing serious economic losses to agriculture production.Moreover,A.lucorum has the habits of polyphagous host-feeding and host transfer.A.lucorum contact complex external chemicals relying on its sensitive olfactory and gustatory system to make the corresponding chemical sensation behavior.This study focused on the chemical sensing function of legs of the A.lucorum in order to further clarify the chemical communication and behavior regulation mechanism of the A.lucorum.1.The sensilla on legs of both males and females are mainly distributed on the meropodium,tibia and tarsus.The sensilla types of different legs of both sexes are the same.There were four sensilla types on legs of A.lucorum by the observation using scanning electron microscopy,sensilla trichodea(subtypes:long straight sensilla trichodea,Str1;long curved sensilla trichodea,Str2),sensilla basiconca(subtypes:medium-long sensilla basiconca,Sba1;short sensilla basiconca,Sba2),sensilla chaetica(subtypes:Sch1,Sch2,and Sch3),and B?hm bristles(BB).The types of sensilla of forelegs,middle legs and hind legs were the same.The distributions of different sensilla types were the same,but the numbers were different.Additionally,the largest number of sensilla was on hind legs whereas the forelegs had the smallest number of sensilla.The TEM results showed that different sensilla contained different number of neurons.2.We sequenced the male and female forelegs,middle legs,and hind legs of A.lucorum using Illumina Hiseq~TMM 2500 platform.There were total 31 chemosensory genes identified using transcriptome data of A.lucorum legs,including 20 odorant binding protein(OBP)genes,eight chemosensory protein(CSP)genes,one odorant receptor(OR)gene,one ionotropic receptor(IR)gene and one sensory neuron membrane protein(SNMP)gene.Compared to the previous antennae transcriptome data,CSP9,10,12,16,17,IR21a and SNMP2a were newly identified in legs.Results of qPCR analysis indicated that all these putative chemosensory genes were ubiquitously expressed in forelegs,middle legs and hind legs of bugs,respectively.The qPCR results showed that OBP9,OBP31,CSP3 were highly expressed in male forelegs,while OBP2,OBP8,OBP17,CSP9,CSP16 were highly expressed in female forelegs.The expression levels of OBP3,OBP11,OBP19 and OBP35 in forelegs as well as middle legs were higher than in hind legs of both sexes.OBP18 was highly expressed in male and female hind legs.Additionally,the expression levels of other genes showed no significant difference among forelegs,middle legs and hind legs of both sexes.3.In vitro recombinant proteins of AlucOBP3 and AlucOBP35 were obtained by cloning,expression and purification.Competitive fluorescence binding assays were performed to analyse the ligand binding characteristics of recombinant AlucOBP3 and AlucOBP35 to 122 candidate compounds.The results showed that the binding substances of AlucOBP3 and Aluc OBP35 were similar.Two recombinant proteins can combine volatile substances and non-volatile substances,and both can combine dimethyl disulfide ether,the component of repellents for the green bugs,suggesting that both are involved in the repellent behavior of the green bugs.Moreover,OBP35 could bound to trans-4-OXO-2-Hexenal(extracted from the body surface of female insect),the component of sex pheromone for the green bugs,indicating that OBP35 might be involved in the near courtship of the A.lucorum.4.Electrolegogram(ELG)tests were performed to investigate the electrophysiological activities with41 candidate ligands.The electrolegogram recordings showed that four odorants,(Z)-3-Hexenyl acetate,trans-2-Hexenyl acetate,(E)-2-Hexenyl butyrate,and trans-2-Hexenoic acid could elicit significant ELG responses to legs.When the standard sample concentration was 0.1?g/?L and 10?g/?L,and the reaction to(E)-2-Hexenyl butyrate,the sex pheromone component of the green bugs,was the largest.When the concentration of odorants was 100?g/?L,the reaction of legs of the green bugs to trans-2-Hexenyl acetate,a volatile substance of a host plant,was the biggest,which further confirmed that the legs of the green bugs were involved in their chemical perception recognition process at the electrophysiological level.
Keywords/Search Tags:Apolygus lucorum, Scanning and transmission electron microscopy, Chemosensory genes, Binding characteristics, Electrolegogram
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