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Identification,Expression And Functional Analysis Of MsepPBPs Of Mythima Separata

Posted on:2021-03-10Degree:DoctorType:Dissertation
Country:ChinaCandidate:X Q ChangFull Text:PDF
GTID:1363330611982866Subject:Agricultural Entomology and Pest Control
Abstract/Summary:PDF Full Text Request
The Oriental armyworm Mythimna separata is a large Noctuidae insect that feeds on Gramineae crops mainly.The fecundity of the moth is as high as 800-1000 eggs per female.Once courtship and mating are successful,the potential harm to crops is very serious.Insects identify all kinds of odor substances in the environment through smell,such as sex pheromones and plant volatiles,so as to determine intraspecific mating objects,habitats or spawning sites.Olfactory function plays an important role in insect behavior.Odor binding protein OBPs/PBPs is thought to play a certain role in the transport of odor molecules in the olfactory recognition process,participating in the initial biochemical recognition process of information chemical perception;PBPs was initially considered to mainly recognize sex pheromone molecules,and PBPs can also improve the binding efficiency between sex pheromones and sex pheromone receptors.However,more and more studies have shown that PBPs can also bind to host plant volatiles and sex pheromone analogues,and some OBPs have been found to bind not only to host plant information chemicals,but also to insect sex pheromone molecules.The physiological function of PBPs in different insects is not very clear.Therefore,on the basis of observing the morphology of antennal olfactory sensor of Oriental armyworm,this paper constructed its antennal transcriptome,analyzed the spatio-temporal expression characteristics of OBPs/PBPs gene,determined the binding characteristics of recombinant PBPs and oriental armyworm sex pheromone molecules,and discussed the function of PBPs gene in the courtship behavior of the oriental armyworm,so as to provide a basis for exploring the physiological function of PBPs in the oriental armyworm.The main results are as follows:1.Morphology of olfactory sensilla on antenna of M.separata.By SEM and TEM photoes,seven morphological sensillum types were recorded in both sexes,including sensilla chaetica,sensilla trichodea(ST I,ST II,ST III),sensilla basiconica(SB I,SB II),sensilla coeloconica(SCo I,SCo II),sensilla styloconica,sensilla squamiformia and B?hm bristles.Based on the presence of poreson the sensillum wall,they could be divided into 3 groups: uniporous sensilla(sensilla chaetica),multiporous sensilla(sensilla trichodea,sensilla basiconica,sensilla coeloconica,sensilla styloconica)and aporous sensilla(sensilla squamiformia and B?hm bristles).S.trichodea were the most abundant sensilla and were distributed over the entire antennae,the wall of S.trichodea was thick and had some pores on it.S.trichodea could be divided into three subtypes,including ST I,ST II,ST III,with female and male dimorphism;the total number of ST I in males was significantly higher than that in females;the appearance of ST I in males was "s",arranged in 3-4rows in clusters,and each segment of sensilla gradually changed from the side of antennae to the middle of ventral surface.The number of ST II and ST III in female was significantly higher than that in male.The wall of S.basiconica was thinner than S.trichodea and had more pores than S.trichodea.The number of SB I in male was significantly higher than that in female,while the number of SB II in male was significantly lower than that in female.The appearance of the S.coeloconica was chrysanthemum like,the number of which in male was significantly higher than that in female.There was no difference in other sensilla between male and female.2.Systematic identification of candidate olfactory related genes of M.separata.130 olfactory candidate genes,including 32 odorant binding protein(OBPs)genes,16 chemosensory protein(CSPs)genes,71 olfactory receptor protein(ORS)genes,8 ionotropic receptor protein(IRS)genes,1gustatory receptor protein(GR)gene and 2 transmembrane protein(SNMPs)genes were identified by sequencing the antenna transcriptome of oriental armyworm.In addition to Msep OBP23,Msep OR1.1,Msep OR3.1 and Msep OR71,126 olfactory related genes have not been identified in the oriental armyworm before.Four candidate genes of sex pheromone binding protein genes(Msep PBPs)in moth were obtained: Msep PBP1(JAV45890.1,Msep OBP23);Msep PBP2 JAV45910.1,Msep OBP3);Msep PBP3(JAV45887.1,Msep OBP26);Msep PBP4(JAV45904.1,Msep OBP9),which all have typical OBPs characteristics and have six conserved cysteine sites.The number,molecular weight and isoelectric point of Msep PBP1-Msep PBP4 were 170,19.1 kd,5.49;165,18.3 kd,5.46;164,18.6 kd,5.46;146,15.7 kd,5.32,respectively.Homology analysis showedthat the identify between Msep PBP1 and Japanese population Msep PBP(BAG71416.1)of the Oriental armyworm reached 98%,and that of Harm PBP(AEB54583.1)was 77%.The identify of Msep PBP3 and Aips PBP(AFM36758.1)was 86%.The identify of Msep PBP4 and cabbage armyworm Mbra PBP(AAL66739.1)was 84%.3.Analysis of temporal and spatial expression characteristics of OBPs/PBPs candidate genes of the oriental armyworm.The expression characteristics of 7 common Msep OBPs(Msep OBP5 ?Msep OBP7?Msep OBP19?Msep OBP20?Msep OBP22?Msep OBP24 ? Msep OBP26)and 4 Msep PBPs(Msep PBP1?Msep PBP4)of the Oriental armyworm were determined by q PCR.The results showed that Msep OBP/PBPs did not show obvious female or male specificity,but had certain temporal and spatial expression specificity.Characteristics of spatial expression: In 7 common Msep OBPs,the expression of Msep OBP5,Msep OBP7,Msep OBP22,Msep OBP24 and Msep OBP26 in antennae was significantly higher than that in head,thorax,abdomen,leg,wing and other tissues,while the expression of Msep OBP20 in antennae was lower than that in other tissues,and Msep OBP19 was widely expressed in all organs,especially in adult wings.Msep PBP1,Msep PBP2 and Msep PBP3 had specific expression in antennae,but Msep PBP4 was slightly expressed in other tissues such as thorax,abdomen,head of mature larvae(including antennae),wings and leg.Characteristics of time expression: There was no significant difference in the expression of the 7 common Msep OBPs except Msep OBP24 on the antennae of adults at different ages.Msep OBP5,Msep OBP7,Msep OBP22,Msep OBP26 in antennae of1-day-old and 3-day-old males was significantly higher than that of other day-old males.The expression of Msep OBP19 in antennae and other tissues of male was also significantly higher than that of other day-old adults;the expression of Msep OBP20 in antennae and other tissues had no significant difference of different age;the expression of Msep OBP26 in antennae of 1-day-old females was significantly lower than that of other day-old females.The expression of Msep PBP1,Msep PBP2 and Msep PBP3 in male was higher than that in other days,which was consistent with thepre-courtship stage of female in 2-3 days,and the expression of Msep PBP4 decreased with the increase of adult age.There was no time-specific expression pattern of 4Msep PBPs in female.4.Cloning,expression and functional analysis of Msep PBPs.The recombinant vector Msep PBPs-p ATX-sumo,was constructed and expressed in prokaryotic system E.coli(EC+).Three recombinant proteins Msep PBP1,Msep PBP3 and Msep PBP4 were obtained successfully.Fluorescence competitive binding test and fluorescence quenching test were used to determine the binding characteristics of Msep PBPs with sex pheromone components of Oriental armyworm,strong male EAG reaction substances and common odor substances.The results showed that the binding of Msep PBPs sex pheromone has a certain specificity.Fluorescence competitive binding test showed that the Ki values of Msep PBP1,Msep PBP3 with sex pheromone components(Z11-16:Ald,16:Ald,Z11-16:OH,Z9-16:Ald,Z11-16:Ac)were 14.32 ?M,10.47 ?M,11.98 ?M,6.84 ?M,13.38 ?M;1.35 ?M,6.30 ?M,5.73 ?M,1.93 ?M,10.94 ?M,respectively.Msep PBP3 has the strongest binding force with the main component of the oriental armyworm sex pheromone Z11-16:Ald.The Ki values of Msep PBP1 and Msep PBP3 with strong male EAG reaction substances((2E,6Z)-nona-2,6-dienal,2-phenylethanol,(E)-non-2-enal)and common odor substances(myrcene,linalool,benzaldehyde,(Z)-hex-3-enal)were 4.96 ?M-27.46 ?M,3.40 ?M-22.55 ?M,respectively.The Ki values of Msep PBP4 with main sex pheromone components were> 50 ?M,while with 16:Ald and Z11-16:Ac were 4.73 ?M and 2.34 ?M,respectively,and the Ki values with strong male EAG reaction substances and common odor substance was 5.48 ?M-8.33 ?M.However,the fluorescence quenching test showed that Msep PBP1 only reacted with Z9-16:Ald,myrcene and(Z)-hex-3-enal,and the slope of Stern-volmer equation showed that Msep PBP1 only bound to Z9-16:Ald statically.Molecular docking showed that Z9-16:Ald bound to Lysine 111 of Msep PBP1 by hydrogen bond.Msep PBP3 only reacted with Z11-16:Ald,Z9-16:Ald,Z11-16 OH,myrcene,(Z)-hex-3-enal,and the slope of Stern-volmer equation showed that Msep PBP3 onlybinded to Z11-16:Ald and Z9-16:Ald statically.Molecular docking showed that Z11-16 Ald bound to Tryptophan 37 of Msep PBP3 by hydrogen bond,and Z9-16:Ald bound to Arginine 110 of Msep PBP3 by hydrogen bond.No compounds had quenching reaction with Msep PBP4.RNAi was used to explore the role of Msep PBP1 and Msep PBP3 which statically bound to sex pheromone in the courtship behavior of the Oriental armyworm.After interference with Msep PBP1,the expression of Msep PBP1 in males was 4.12% of that of the control,and after interference with Msep PBP3,the expression of Msep PBP3 in males was 53.48% of that of the control.Through the behavior observation platform,it was found that the standing time before the first flight of these two interfered males were 498.67±286.21 min,490.47±214.87 min respectively,which was significantly longer than that of the control 14.95 ±7.97 min.100% of the control males showed flight behavior,20% of the males flew but did not mate,80% of the males did not fly and mate;20% of the males did not fly after Msep PBP1 interference,60% of the males flew but did not mate,20% of the males did not fly and mate;after Msep PBP3 interference,5% of the males did not fly,80%of the males flew but did not mate,and 15% of the males flew and mated.The results showed that after the interference of Msep PBP1 and Msep PBP3,the sensitivity of males to females decreased,and it was directly proved that Msep PBP1 and Msep PBP3 could improve sensitivity to the sex pheromones molecule.In summary,the experiments showed that Msep PBP3 may be the key protein of the reception of sex pheromone molecules of the Oriental armyworm,and Msep PBP1 and Msep PBP3 had certain specificity to the sex pheromone molecules,and these two proteins may play an important role in improving the sensitivity of sex pheromone molecules of the moth.These results provide a theoretical reference for an in-depth understanding of the molecular mechanism of insect recognition of sex pheromones and the field application of sex pheromones,and provide data support for further interpretation of the function of PBPs of the Oriental armyworm.
Keywords/Search Tags:The oriental armyworm, Antennae, Olfactory sensor, Fluorescence competitive binding, Fluorescence quenching, RNAi, Behavior
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