Study On Pheromone Binding Proteins And Active Components Of Contact Pheromone In Liposcelis Entomophila

Insects use complex and sensitive olfactory system to accurately identify odor substances in habitats,seeing advantages and avoid disadvantages,and then complete physiological activities such as foraging,oviposition and avoidance of natural enemies.Chemosensory proteins(CSPs)are an indispensable part of insect olfactory system,which can recognize host plant volatiles and pheromones.Booklice,also known as rice lice,belong to Rodentae,Fenderrodentae,Narodentia,Lycopodidae and Lycopodidae.It is a very small insect with a body size of only 0.6-1.5 mm.Booklice widely exist in the world in the past 20 years.Booklice have gradually become to the dominant population of stored grain pests.Finding a reasonable way to control booklices is a great challenge to gurantee grain safety.Biological control methods have been effectively used in the control of some insects,but there are few studies on the biological control of booklice.Therefore,from the perspective of molecular biology and chemical ecology,it is of great significance to systematically explore the mechanism of chemoreceptor proteins in the prevention and control of the Liposcelis entomophila.Firstly,based on the data of transcriptome sequencing of L.entomophila,the present study was obtained and analyzed the open reading frame sequence of chemonsensory protein gene Liposcelis entomophila CSP in L.entomophila.Real-time PCR was used to analyze expression pattern of LeCSP gene between antennae of male and female adults and in different developmental stages.The results showed LeCSP(Genbank accession NO.MK211168)was cloned and identified as the chemosensory protein gene of L.entomophila.The open reading frame length of LeCSP was 411 bp,encoding 136 amino acids.The predicted molecular weight was 17.45 kDa and the isoelectric point was 9.45.LeCSP has a complete signal peptide located at 1-18 amino acids.There were four conserved cysteine residues in the amino acid sequence of LeCSP,which were 56.93%and 54.474%homologous to Tribolium castaneum and Tenebrio molitor,respectively.qPCR results showed that the expression of LeCSP in male antennae was significantly higher than that in males.The sequence of LeCSP was cloned and the expression of LeCSP gene in the antennae of adult worms was clarified,which laid a foundation for further study of its function and better understanding of the role of LeCSP gene in olfactory recognition of L.entomophila.The expression vector was successfully constructed by prokaryotic expression.The expression of LeCSP protein in supernatant was detected by inducing the expression of recombinant plasmid and SDS-PAGE analysis.The recombinant protein LeCSP was purified by Ni-NTA His Bind Resin column.The mass concentration of the recombinant protein LeCSP was 1 mg/mL.Using 1-NPN as a fluorescent probe,the binding ability of recombinant protein to volatiles and sex pheromone analogues of the main host of the insect-loving bookfly was tested by fluorescence competitive binding assay.The binding constant(K_d)of LeCSP was6.351?M.Among them,the protein has strong binding ability to 1-octene-3-ol,hexadecanoic acid,oleic acid,linoleic acid and vanillin,which proves the ability of LeCSP protein to bind to phagemidin analogs.And identified these five substances as the main components of the pheromone contact pheromone.Odor selection experiments in laboratory were carried out with“Y”-type olfactometer to test the selectivity of mature male insects to five reported sex pheromone analogues of the L.entomophila.The attracting effect of a single compound on the L.entomophila was clarified,which provided a basis for the development of attractants.It provides a basis for the development of food attractants.At the same time,two kinds of attractants made according to specific components were tested in a warehouse in Zhengzhou City.The results of odor selection test showed that all the five attractants had trap effects on the tested insect.There were significant differences in the trap effects under different concentrations.There were certain effective ranges of different volatiles on the action concentration of the L.entomophila,and there were also certain differences in the action concentration of different volatiles.The results of field trapping experiment showed that the combination of two kinds of attractants and conical traps had significant trapping effect on the L.entomophila.The results could be use to predict the occurrence of the L.entomophila for a long time,and also forecast the occurrence location of the L.entomophila in grain heap.