Cloning And Analysis Of Chemosensory Protein Genes In Monochamus Alternatus Hope

Monochamus alternatus(Hope)commonly known as pine sawyer beetle,considered an important pest in forestry.It not only causes harm to trees,but also it is the main vector of pine wood nematode disease.Biological control,based on behavior regulation,is an important means to control pine and pine wood nematode disease.Insect antennae are the main organ of the insect to play the olfactory function.The antennae produce various olfactory sensors,and are the basic unit for insect identification of various environmental signals.Chemosensory protein(CSPs)is a key agent in insect antennae that specifically binds and transport odor molecules.It plays an important role in insect recognition of information chemical substances.It will provide a theoretical basis for better biological control if the chemosensory mechanism of pines can be clearly defined.However,the specific function of the chemoreceptor of Pinus Brown is still not clear.So we use Monochamus alternatus Hope as the research object,observe the ultrastructure of the antennal sensilla,and cloned a chemosensory protein MaltCSP5,analyzed the spatio-temporal expression of MaltCSP5 gene recombinant MaltCSP5 fluorescence spectrum,and 13 kinds of pine volatiles were determined with characteristics.The main results are as follows:1.Antennal sensilla of M.alternatus and its ultrastructureWe observed male and female receptors in mature individuals with the help of scanning electron and transmission electron microscopy.Various kinds of sensilla(sensilla basiconica,sensilla trichodea,sensilla digit-like,sensilla rod-like,and sensilla bottle-like)were seen on the male's antennae while on the female antennae,there were four kind such as the sensilla basiconica,sensilla trichodea,sensilla bottle-like,and sensilla chaetica.The sensilla basiconica and trichodea were abundantly present on the front elevation of the antennae.The transmission electron micrscropy results of sensilla basiconica on the antennae of insect depicted that the cuticle has no holes.Results of longitudinal sections of sensilla trichodea exhibied them to be with smooth surface and have flat tips while the pegs are pointed tipped that are suggest to be responsible for gustatory function.Micrographs of transmission electron mircroscopy of the longitudicnal sections of sensilla chaetica clearly exhibits dendrites without cuticular pores;suggesting that these sensilla might serve in the perception of heat,temperature and humidity,and carbon dioxide.2.Temporal and spatial expression and binding characteristics of Malt CSP5RT-PCR technique was successfully cloned to clone MaltCSP5.The open reading frame was 390 base pairs,encoding 129 amino acids,encoding the relative molecular mass 15KDa of the protein,containing 4 conserved cysteine.Results of the Quantitative reverse transcription PCR(RT-qPCR)showed the expression of the CSP5 gene in male and female antennae.At the same time,recombinant expression plasmid was successfully constructed and recombinant protein was obtained by affinity chromatography.Competitive binding assays were performed using 1-NPN as a fluorescent probe to determine the binding affinity of recombinant CSP5 to 13 odour molecules of pine volatiles.Results exhibited that CSP5 showed very strong binding abilities to myrcene,(+)-?-pinene and(-)-isolongifolene at pH7.4,whereas the volatiles 2-methoxy-4-vinylphenol,p-cymene and(+)-limonene oxide have relatively weak binding affinity at pH 5.0.