| Cyrtotrachelus buqueti(Coleoptera: Weevils)is a dried moth pest that pests bamboo plants,which occurs in the southern cultivation area of bamboo in China,which seriously restricts the development of the bamboo industry,but the insect is also a resource insect that can be viewed and eaten.At present,the prevention and control measures for the Cyrtotrachelus buqueti is mainly based on chemical control and artificial capture,not only the prevention and control effect is not good,but also the chemical control is easy to pollute the environment and produce pharmaceutical harm,and can not be turned into benefits,to achieve the resource management of the Cyrtotrachelus buqueti.Insects have developed olfactory receptors,which can identify a variety of signal substance in the external environment to seek mate foraging.Sensory neuron membrane proteins SNMPs are an important class of olfactory sensing related proteins in insect olfactory receptors,which play an important role in the process of insects recognizing external odor substances.Therefore,in-depth study of the function of SNMPs has far-reaching significance for understanding the olfactory receptor mechanism of insects and thus greening the control of pests.Based on the transcriptome database(Gen Banknumber:SAMN06176790),a sensory neuron membrane protein gene Cbuq SNMP1 b was cloned and the phylogenetic relationship of the gene was analyzed;the expression of Cbuq SNMP1 b in different tissue sites and developmental stages of Cyrtotrachelus buqueti was analyzed by real-time fluorescence quantitative q PCR technology,and the prokaryotic expression and purification of Cbuq SNMP1 b were completed.The homologous modeling of Cbuq SNMP1 b and the molecular docking of Cbuq SNMP1 b with sex pheromone-binding proteins and odor molecules were carried out by Discovery Studio2019 software,the key binding sites and binding forces were predicted,and the interaction between Cbuq SNMP1 b and Cbuq PBP2 was verified by yeast double hybridization experiments,and the main results were as follows:1.Based on transcriptome data and cloned by RT-PCR technology,the sensory neuron membrane protein gene Cbuq SNMP1b(Gen Bank number: OM908752)was obtained.It has an open reading frame of 1605 bp,encoding 534 amino acids,a protein molecular weight of 60.96 k Da,and a theoretical isoelectric point of 8.30.Cbuq SNMP1 b is a class of alkaline hydrophilic proteins with typical features of the CD36 protein family,N-terminal signalless peptides,containing two transmembrane domains and a macrocyclic outer ring containing six conserved cysteine residues.The results of the phylogenetic tree show that Cbuq SNMP1 b belongs to the SNMP1 subfamily,and has a high homology with insects of the Coleoptera Weevils,62.07%to 79.17%,and is also closely related.2.QPCR results showed that in different tissue sites,CbuqSNMP1 b was expressed the highest in adult antennae,less in the feet and wings,and the antennae were significantly different from other parts,and the males were higher than the females;at different developmental stages,Cbuq SNMP1 b was expressed the highest in adults,less in the pupal stage,weak in the mature larval stage(5 instar),almost no expression in the young larval stage(2nd instar)and the egg stage,significantly different in adults and other stages,and the males were higher than the females.It is speculated that CbuqSNMP1 b may play a role in the process of foraging for males.3.Successfuly constructed prokaryotic expression plasmid CbuqSNMP1b-pET-28a(+),introduced into E.coli Rosetta(DE3)competent cells,and successfully expressed Cbuq SNMP1 b recombinant protein after induction,SDS-PAGE showed that it was mainly expressed in inclusion bodies,and purified to obtain a recombinant protein of about 51 k Da.The purified protein was subjected to fluorescence competition binding experiments with 15 odorous substances,and the results showed that Cbuq SNMP1 b could effectively bind to 4 kinds of insect volatiles.Among them,CbuqSNMP1 b has the strongest binding ability to dibutyl phthalate(DBP),with a dissociation constant of 9.03 μmol/L;Cbuq SNMP1 b has a strong binding ability with bis(2-ethylhexyl)phthalate(DOP)and benzothiazole,with dissociation constants of 15.53 μmol/L and11.59 μmol/L,respectively,and Cbuq SNMP1 b having a weak binding capacity with phenol,with dissociation constant of 20.95 μmol/L.4.The Cbuq SNMP1 b model was constructed using Discovery Studio2019 software,which has six conserved cysteine residues(CYS279,CYS308,CYS344,CYS346,CYS352,CYS363),forming three pairs of disulfide bonds(CYS279-CYS344,CYS308-CYS363,CYS346-CYS352).The protein-protein molecular docking results show that the main driving force for the formation of the complex is van der Waals force.The hydrophobic(non-polar)ratio between the two complexes is 48.01% and45.03%,respectively,and there are more hydrophobic interfaces,which can maintain structural stability;the main force of the binding interface of the Cbuq SNMP1b-Cbuq PBP1 complex is 7 short strong hydrogen bonds,and the Cbuq SNMP1b-Cbuq PBP2 complex is 9short strong hydrogen bonds.The Cbuq SNMP1b-Cbuq PBP2 complex has stronger force and more stable spatial structure.The protein-odor molecular docking results showed that Cbuq SNMP1 b combined with DBP to form two short strong hydrogen bonds,one short strong hydrogen bond with DOP,and no short strong hydrogen bond was formed with ethyl caproate and benzothiazole.Therefore,Cbuq SNMP1 b has better binding ability with DOP and DBP,and the complex formed is more stable,and it is speculated that Cbuq SNMP1 b may play a role in the process of odor molecule recognition.5.The interaction between Cbuq SNMP1 b and CbuqPBP2 was studied by yeast two-hybrid system,and after bait plasmid toxicity detection,self-activation detection and functional verification,the bait recombinant plasmid SNMP-p BT3-N and the prey recombinant plasmid PBP2-p PR3-N were co-transformed into NMY51 yeast strain,and TDO/3’AT 5m M and QDO plates were found by plating no colonies were grown,indicating that there is no interaction between SNMP-p BT3-N and PBP2-p PR3-N in the current validation systems,and that there was no binding or weak binding force between CbuqSNMP1 b and CbuqPBP2. |
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