Functional Study Of Pheromone Binding Proteins In Yellow Peach Moth Conogethes Punctiferalis(Guenée)

As a harmful insect,Conogethes punctiferalis(Guenée)caused serious economic damage to crops and fruit trees in China.Due to the concealment of its larvae,extensive use of chemical pesticides caused a variety of potential threats in the ecological environment.The communication between insects and the environment rely on olfactory system,which plays a crucial role in insect behaviors,such as food and host seeking,mating,ovipositing and escaping adverse environment.Therefore,the study of olfactory mechanism can provide us new biological methods to control this pest through interring their olfaction perception.The main contents and results of this study are as follows:1.The binding experiment results showed that the CpunPBP1 protein had a good binding ability with the most tested compounds,and had the strongest binding ability to the sex pheromone.Among the plant volatiles,CpunPBP1 had strong binding ability to 1-tetrodecanol,trans-caryopyllene,farnesene and ?-farnesene.Cpun PBP2 had the strongest binding ability to sex pheromone,followed by minor pheromone hexadecanal.Among the plant volatiles,CpunPBP2 had a better binding abiliy to ?-farnesene,farnesene,trans-caryopyllene.CpunPBP3 showed a weak combination with sex pheromones and plant volatiles,among the plant volatiles,CpunPBP3 only showed a weak binding ability to ?-farnesene,linalool and palmitic acid.2.The expression levels of CpunPBP1,CpunPBP2 and CpunPBP3 in the tissues of the female and male moths were determined by RT-q PCR,and the distribution in antennal sensilla were measured by immunohistochemical method.The results of qPCR showed that all the three PBPs were mainly expressed in antennae of both sexes,with low expression in other tissues.The expression of CpunPBP1 in antennae of female was significantly higher than in the male,the CpunPBP2 was male-specific,and expression level of CpunPBP3 in male and female antennae is similar.The results of immunohistochemical experiments showed that the three proteins were mainly labeled in trichoid sensilla,and CpunPBP2 and CpunPBP3 were also labeled in bascionic sensilla,which is sensitive to the plant volatiles.It is note worthy that not all trichoid sensilla are labeled with the CpunPBP1,CpunPBP2 and CpunPBP3.Only a few trichoid sensilla in the female was labeled,indicating that the expression of the three proteins in the female antennae was low.Other sensillas such as the coeloconic sensilla and chaeticum sensilla were not labeled,indicating that the three proteins may not be expressed in these sensillas.3.The crystal structure of Amyelois transitella and Bombys mori PBPs were chosen as template for CpunPBP1 and CpunPBP2 to predict the three-dedimensional structures,respectively.Furthermore,based on the strong binding affinity,homology modeling and molecular docking of CpunPBP1 and CpunPBP2 with sex pheromones E10-16: Ald and Z10-16: Ald were conducted to understand the structural basis.The binding sites of CpunPBP1 and CpunPBP2 were predicted according to the hydrophobic interactions.Finally,Ser9,Phe12,Val115 and Arg120 were predicted for CpunPBP1 as key binding sites,and Phe9,Phe33,Ser53 and Phe115 were predicted for Cpun PBP2.The binding affinities of four mutants of CpunPBP1 and four mutants of CpunPBP2 with the two sex pheromones were investigated by fluorescence competitive binding assays.Compared with CpunPBP1,the binding ability of all CpunPBP1 mutants to sex pheromones were reduced in different degree.Compared with CpunPBP2,the mutant Cpun2-m4 likely lost the binding ability to the two sex pheromones.The binding abilities of remaining three mutants showed no significant differences with wild CpunPBP2.4.Based on the observation of mating behavior,the EAG technique and wind tunnel experiment were used to study the electrophysiological and behavioral studies of C.punctiferalis.The results were as follows: The mating rhythm had a difference between population mating and sing mating.Totally,the mating peak mainly focused on 22:00 to 0:00 of the second and third day.EAG results showed that the male antennae had a higher responses to pheromones than odorants.Among the odorants,the unmated male antennae had a high response to benzaldehyde and cis-3-hexen-1-ol,while the mated male antennae had a high response to trans-2-hexenal and cis-3-hexenyl-acetate.The virgin and mated female moth have no response to pheromone.The response to trans-2-hexenal stronger than other odorants,and the response of the mated female antennae to hexenal,?-pinene,benzaldehyde and trans-2-hexenal were significantly lower than virgin female antennae.In the wind tunnel experiment,the attractive ability of pheromone(Z:E=1:9)in five stages(take off,oriented upwind fight,arrival in the middle of tunnel,close to source(10 cm)and touch down source)were significantly higher than others lures,and benzaldehyde can significantly enhance the attraction of pheromone(Z:E=1:9)to male.5.Using Illumina sequencing platform and bioinformatics analysis,we identified several olfactory gene families from Conogethes punctiferalis antennal transcriptome.The expression levels of ORs are evaluated by qPCR.The main results were as follows:(1)A total of 20 OBPs genes were annotated.FPKM analysis showed that 10 OBP genes(CpunGOBP2,CpunOBP6,CpunGOBP1,CpunPBP4,CpunOBP17,CpunOBP13,CpunOBP12,CpunOBP16,CpunPBP1,CpunOBP2)(FPKM> 1000)mostly expressed in male and female antennae.(2)13 CSPs genes were identified.FPKM values showed that CpunCSP6,CpunCSP1,CpunCSP11,CpunCSP2,CpunCSP14 were higher expressed in antenne(FPKM> 1000).(3)83 CRs genes including 62 ORs gene,11 IRs gene and 10 GRs genes were screened.FPKM analysis showed that the expression of olfactory receptor co-receptor CpunOrco in antennae was highest than other ORs.The remaining ORs?IR and GR genes were low expressed in antennae.(4)Two SNMPs were identified from antrnnal transcriptome.CpunSNMP1 and CpunSNMP2 had open reading frame with 527 and 523 amino acids,respectively.The FPKM values showed that the expression of these two genes was similar in male and female antennae.(5)The results of RT-qPCR showed that OR8,OR39,OR48,OR4 and OR30 were mainly expressed in female antennae.Pheromone receptors OR1,OR3,OR5 and OR7,as well as general olfactory receptors OR58 and OR62 were mainly expressed in male antennae.