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Olfactory Genes Screening And Function Analysis Of Pheromone Binding Proteins Of Eogystia Hippophaecolus(Lepidoptera:Cossidae)

Posted on:2018-11-18Degree:DoctorType:Dissertation
Country:ChinaCandidate:P HuFull Text:PDF
GTID:1363330575991596Subject:Forest Protection
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Seabuckthorn carpenterworm Eogystia hippophaecolus(Hua et al.)(Lepidoptera:Cossidae)damages the ecological economy tree seabuckthorn Hippophae rhamnoides L.(Rosales:Elaeagnaceae),which is widely distributed throughout "Three North" protection forest in China and prevents soil erosion and desertification.Owing to its complex ecological and life history traits,for example,the larvae bore into trunks and roots,and complete one generation every 3-4 years,a highly effective method to control the damaging larvae has not been developed,which caused enormous ecological and economic losses in China.By now,by extracting female sex pheromone glands,identifying extracts,electroantennographic(EAG)analyses,and field trials,the sex pheromones of E.hippophaecolus female sex pheromone gland have been identified as(Z)-7-tetradecenyl acetate(Z7-14:Ac),(E)-7-tetradecenyl acetate(E7-14:Ac)and(E)-3-tetradecenyl acetate(E3-14:Ac),and have been used to develop specific and efficient artificial sex pheromone traps,which monitoring and trap effect is obvious.But we don't know the molecular mechanism of male E.hippophaecolus identification sex pheromone.Identification of olfactory related genes and function study of E.hippophaecolus will help us to clarify the molecular mechanism of insect olfaction recognition,providing theoretical basis for development new technology to prevention and control pest.In this research,based on the male and female antennae transcriptome of E.hippophaecolus,selected a series of olfactory related genes;knew explicit expression characteristics of odorant binding proteins;aiming at phenomenon of male specific recognition of sex pheromone components,explored the sex pheromone binding proteins(PBPs)function from the transcription and protein levels.The main research results are as follows:1.Pinpointed the sensillum quantity,type,and distribution of four olfactory functions organs in E.hippophaecolus(the following called "four organs").Sensillum on E.hippophaecolus male antenna was two types of sensillum trichodea,two types of sensillum basiconica,a type of sensillum coeloconica,and bohm's bristles.The labial palps had sensillum trichodea and sensillum chaetica.On the external genitals,three types of sensillum trichodea were the only sensillum.On the legs(propodeum,mesopodium,and metapedes),the most sensillum,sensillum trichodea,and sensilla chaetica were on the tarsus.Among the sensillum on four organs of E.hippophaecolus,the main chemosensilla are sensillum trichodea,sensillum basiconica and sensillum coeloconica,which quantitative relation in olfactory organs was antenna>external genitals>labial palps>leg.2.Identified 137 olfactory proteins through males and females antennae transcriptome of E.hippophaecolus.Included 29 odorant binding protein(OBPs),18 chemosensory proteins(CSPs),63 odorant receptors(ORs),12 ionotropic receptors(IRs),13 gustatory receptors(GRs)and two sensory neuron membrane proteins(SNMPs).Among 29 odorant binding proteins contained 3 pheromone binding proteins(PBPs)and 2 general odorant binding protein(GOBPs).3.Pinpointed Odorant binding protein(OBPs)expression profile in four organs of male E.hippophaecolus.Most OBPs were bias expressed in antenna,indicating most OBPs' function as participating in the antennal olfactory recognition.Few OBPs bias expressed in the leg and external genitals,showed leg and external genitals also played a considerable role in odor identification,which speculed OBPs in leg and external genitals helped identify the host volatiles and assisted in recognition of sex pheromone components.4.Pinpointed three PBPs' expression profile in four organs of male and female E.hippophaecolus,PBPs were bias expressed in antenna,especially in male antenna which was extremely obvious expression.In male antenna,PBP1 expressed highest,which was 10 times of PBP3 expression quantity and 1700 times of PBP2 respectively,speculated PBP1 and PBP3 were main proteins that participated in identifying sex pheromone in antenna.In addition to antenna,PBPs highest expressed in leg,especially in male leg,and male external genitals expressed PBPs,combined with mating behaviour of seabuckthorn carpenterworm,speculated that leg and male external genitals can identify pheromone and help mating,but female external genitals not.5.Pinpointed three PBPs' protein expressions in four organs of male and female E.hippophaecolus.Through cloning we got complete ORF of three pheromone binding protein genes.Sequence alignment and 3D structure analysis showed that PBP1,PBP2 and PBP3 had structure characteristics of pheromone binding proteins.Recombinant expression of PBP1-3 of E.hippophaecolus all existed in inclusion body.Through crushing,renaturation and purification,there was single band at about 16 kDa.Western Blot results almost accorded with mRNA level.Expression organ was that PBP1 expressed in male antennae,leg,external genitals,labial palps,also in female antennae and leg;PBP2 only expressed in antenna;PBP3 expressed in male antennae and leg,and female antennae.The expression quantitative relationship was that antenna was the highest expression organ of PBP1-3,male antennae more than female,and PBP1 expression quantity bigger than PBP3,PBP3 bigger than PBP2,which supported that male antennae was major PBPs expression organs of E.hippophaecolus and PBP1 and PBP3 were main protein participating in identifying sex pheromone protein.Besides,except antenna,PBP1 and PBP3 highest expressed in leg,as well as male external genitalia expressed PBPs,which also supported that the male leg and external genitals of E.hippophaecolus assisted the process pheromone recognition.6.Pinpointed binding ability to sex pheromone,analogue and plant volatile among pheromone binding proteins PBP1-3 of E.hippophaecolus.PBP1 and PBP3 binding with two sex pheromone components strong,and PBP1 detection ability of sex pheromone components was stronger than PBP3.Combination with sex pheromone analogues showed that PBP1 and PBP3 cannot distinguish the carbon double bond location,isomer and residues difference of 14 carbon sex pheromone analogues.PBP2 can only appropriate distinguish difference between carbon double bond position and isomer.Combination with plant volatiles showed that PBP1 can also help to identify the host.All in all,antenna,especially male antenna was major organ to sex pheromone recognition of E.hippophaecolus,besides male leg and external genitals of E.hippophaecolus assisted the process pheromone recognition.PBPlwas highest expressed in antenna,especially in male antenna obviously higher than female,and its strongest combination ability with sex pheromone,so which was the main protein to recognition of sex pheromone component.At the same times,it also combination seabuckthorn volatiles,so it can help seabuckthorn carpenterworm to identify host.PBP3 was high expressed in antenna,especially in male antenna obviously higher than female,and with strong combination ability with sex pheromone,weak binding with seabuckthorn volatiles,so which was another protein to recognition of sex pheromone largely.Expression and combination ability of PBP2 was low and weak,and can properly distinguish carbon double bond location and isomer,speculated that PBP2 contributes to the specificity of sex pheromones recongnization.
Keywords/Search Tags:Eogystia hippophaecolus, pheromone binding proteins, antennal transcriptome, prokaryotic expression, expression profile, binging ability, Western blot
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