Functional Analysis Of Odorant Binding Proteins And Chemosensory Proteins From Monochamus Alternatus Hope

Monochamus alternatus Hope is a key pest on pine trees,not only for its own damages like boring and feeding,but also for its transmission of Bursaphelenchus xylophilus.Olfactory play key role in host location,mating and oviposition of M.alternatus.Previous researches have found that small,soluble proteins like odorant binding proteins,chemosensory proteins played important roles in the first step of olfactory recogniaton in insects,thus a deeper understanding of its molecular mechanism will be very helpful in control of M.alternatus Hope and pine wood nematode disease.With this purpose we carried out experiments described as below:1.Immunolocalization of MaltOBPs in the antennal sensillaTypes of antennal sensilla were examined using scanning electron microscope?SEM?.Rabbits were injected with purified MaltOBPs?MaltOBP9,MaltOBP10,MaltOBP19,MaltOBP24?to make polyclonal antibodies.Western blotting analysis suggested that both anti-MaltOBP9 and anti-MaltOBP10 may recognize their targets from the total proteins of male and female antenna.Immunocytochemical labelling results showed that MaltOBP9 and MaltOBP10 were all expressed in the sensillum lymph of Sch,Str,Sb,and Sa.Which suggested that MaltOBP9 and MaltOBP10 might involved in recognition of general odors and pheromones.2.Expression profiles of different MaltOBPs genes and RNA interference effect detectionGene expressions of MaltOBPs in antennae of male and female M.alternatus were tested at different stages of development through quantitative real-time PCR.No distinct regularities were found.By and large,these genes were expressed highly on 6-9th days aftrer copulation.In that case,5-days-old adults were chosen to investigate the RNA interference effect.The results revealed that gene expression decreased from the 3-4th days after injection,even though the interference effect was not significant.3.Expression,purification of MaltCSPs and binding affinities assaysRecombinant plasmids pET30a-MaltCSP7 and pET32b-MaltCSP8 were successfully constructed,pET30a-MaltCSP7 mainly expressed in supernatant,while pET32b-MaltCSP8 mainly expressed in inclusion bodies.Finally pET32b-MaltCSP8 was purified through Ni sepharose high performanc column.The fluorescence competitive binding assay revealed that MaltCSP8 showed higher binding properties for all 16 host volatiles in pH 7.4 than in pH 5.0.It particularly highly combined with?+?-limonene oxide,?+?-?-pinene,?-terpinene and myrcene in pH 7.4(K₄)<10).The fluorescence quenching assay of MaltCSP8 with these 16 host volatiles in pH 7.4 all indicated a static quenching mechanism.The apparent association constant and binding sites suggested that MaltCSP8 had stronger binding abilities in neutral environment.Thermodynamic parameters such as the free energy change,enthalpy change,and entropy change implied that the acting forces between MaltCSP8 and host volatiles mainly originated from Hydrogen bonds and Van der Waals forces.4.Behavioral responses of male and female M.alternatus to several volatilesBased on the binding data,several volatiles were chosen to confirm if they have effects on the adults of M.alternatus Hope using Y-tube test.Liquid paraffin and odor of1%concentration?diluted in liquid paraffin?were placed at the two arms as attractants.Results showed that 1%?1R?-?+?-?-pinene may attract female adults,whereas the male adults revealed phobotaxis to 1%?-terpinene and myrcene.