Functional Studies On Odorant Binding Protein MmedOBP18 And Odorant Receptor MmedOR19,25,48 Of The Microplitis Mediator Haliday

Microplitis mediator Haliday(M.mediator)belongs to Hymenoptera,Braconidae and the species were first recorded in China in 1979.M.mediator was a broad-spectrum endoparasitic parasitic wasp,which could parasitize the young larva stage of Lepidoptera pests to achieve the biological control effect,such as Helicoverpa armigera Hubner and Mythimna separata Walker This experiment mainly studied the location and function of the odorant binding protein MmedOBP18 and odorant receptors MmedOR19,MmedOR25 and MmedORA8 that were highly expressed in the antennae,separately.Fluorescence revealed that MmedOBP18 had in the cone-type sensor I with the antennae of the female M.mediator and it was speculated that it had the function of the short-range recognition and taste recognition function.The function of the potential ligand was determined by the fluorescence competitive binding experiment.For the gene of MmedOR,through in-situ hybridization,MmedOR19 was located in male antennae,MmedOR25 and MmedOR48 were located in female antennae,which corresponding ligands were identified by two electrode-voltage clamp.The main function of MmedOR19,MmedOR25 and MrnedOR48 was to identify the host or host habitat.Finally,by EAG technology,the identified volatile ligands were tested by male and female antennas.In summary,this experiment located odorant binding protein MmedOBP18 and MmedOR19,MmedOR25,MmedOR48 respectively in the antennae.The fluorescence competitive binding experiment for MmedOBP18 and voltage clamp experiment were used to screen the ligands for MmedOR19,25,48 to explore functions,which made sure the important roles of these four proteins in chemical perception of the Microplitis mediator.1.The odorant binding protein MmedOBP 18 was located on the antennae cone-type sensor I of the M.mediator.Using fluorescent immunohistochemistry,the distribution of MmedOBP18 in the antennae of the M.mediator was determined by protein localization.It could be observed that there was a large amount of aggregation in cone-type sensor I,which was suggested that its function might be related to taste.2.Three odorant receptors were localized in the antennae of the M.mediator.Through in-situ hybridization,biotin was linked to antenna tissue by RNA probes for fluorescence labeling.It concluded that odorant receptors might play an important role in the identification of chemical communication information of the M.mediator.MmedOR19,MmedOR25 and MmedOR48 were located in the antennae of the M.mediator.3.The recognition characteristics of ligand binding of odorant binding protein MmedOBP18 were elucidated.For MmedOBP18 ligand binding characteristics,the ligands were obtained from the fluorescence competitive binding experiment showed that low volatile compounds including undecylic acid(Ki=6.58±0.02 μmol/L),lauric acid(Ki=17.69±0.05 μmol/L),tridecanoic acid(Ki=11.68±0.08 μmol/L),myristic acid(Ki=6.49±0.03 μmol/L),dodecyl aldehyde(Ki=6.42±0.03 μmol/L),decanal(Ki=15.73±0.05 μmol/L),2-undecanone(Ki=10.36±0.06 μmol/L),2-tridecanone(Ki=5.21±0.02 μmol/L),decanol(Ki=11.63±0.07 μmol/L),octane(Ki=14.37±0.04 μmol.L),lepidopteran pheromone components(Z)-9-tetradecenal(Ki=9.09±0.06 μmol/L)and(Z)-11-hexadecenal(Ki=11.67±0.03 μmol/L)were strongly binded with recombinant MmedOBP18.It was also found that some non-volatile plant secondary metabolites,such as linolic acid(Ki=6.51±0.03 μmol/L),palmitic acid(Ki=3.86±0.02 μmol/L),quercetin(Ki=5.08±0.02 μmol/L)and gossypol(Ki=5.07±0.01 μmol/L)were strongly binded with recombinant MmedOBP18.The function of the MmedOBP18 for the above semi-volatiles and non-volatiles further illustrated its existing taste and smell recognition function,which had a profound influence on the study of the chemical communication mechanism of the host recognition of the M.mediator.4.Explaining the characteristics of odor ligand recognition of three kinds of M.mediator odorant receptors.In vitro eukaryotic expression of the odorant receptor of the M.mediator.In vitro eukaryotic expression was performed by injecting clam frog egg cells into reverse transcriptome the cRNA.Only 20±4.3 nA was identified for octanal of the MmedOR25,while 48±1.8 nA and 20±7.5 nA were identified the reaction of nonanal and octanal for the gene of MmedOR19.However,the response of MmedOR48 was very obvious and showed a wide spectrum of odor ligands.MmedOR48 was mainly used for the recognition of alcohol and aldehyde in plant volatiles.The response of odor ligands were mainly identified that were octanal,nonanal and decanal,which the currents were 296±61.3 nA,509±107.7 nA and 424±85.1 nA,respectively.Using different concentrations from 10-8mmol/L,10-7mmol/L,10-6mmol/L,5×10-6mmol/L,10-5mmol/L,5×10-5mmol/L,10-4mmol/L,5×10-4mmol/L to 10-3mmol/L,which was found that the concentration increased with the concentration and suggested its function for the identification of host habitat chemical information.5.To verify the identification characteristics of the M.mediator on odor ligands.Preliminary screening of EAG to the odor ligands of the M.mediator.Through EAG technology,we tested and verified the ligand of the M.mediator,including decal,octanal,dodecal,undecal,heptanal,nonaldehyde,maleic aldehyde,2-undecanone,2-undecanone,octane,red myrrh,decanol,undecanoic acid,dodecanoic acid,and lepidopterin(Z)-9-tetradecenal.The results showed that the ligands above indeed played an important role in the identification of chemical information of the M.mediator.At the same time,the co-recognition ligand of MmedOBP 18 and MmedOR19,MmedOR25 and MmedOR48 also indicated that both odorant binding protein(OBP)and odorant receptor(OR)played an critical role in the chemical communication of the M.mediator.