| Agrilus zanthoxylum,a destructive borer of Zanthoxylum bungeanum,was first reported in 1988.However,due to its hidden symptoms,physical control and chemical control with single application had little effect,which makd it difficult to prevent and control.Olfactory recognition mechanism is very important for host identification,but it has not been fully understood in this pest.Odor-binding protein plays an irreplaceable role in the life process of the localized host of A.zanthoxylum.As the first reaction in the olfactory recognition process of insects,odor-binding protein was the first to capture hydrophobic odor molecules and transfered them to sensory lymph.In this study,three odor-binding proteins of A.zanthoxylum were expressed,purified and predicted by bioinformatics and molecular biology methods,and the binding characteristics between odor-binding protein AzanOBPs and host volatiles were analyzed.The main results were follows:1.The full-length sequences of three odor binding protein genes(AzanOBP4,AzanOBP5 and AzanOBP7)of A.zanthoxylum were cloned,and the 5’ and 3’ non-coding regions were identified.According to bioinformatics analysis,it was found that the amino Agrilus mali acid sequences of the three genes had high homology with the odor binding protein,among which AzanOBP4 and AzanOBP5 belonged to the Classic OBP family,and AzanOBP7 belonged to the Minus-C OBP family.2.All three genes expressed in different tissues(head,thorax,abdomen,feet and wings)of A.zanthoxylum,AzanOBP7 with the highest relative expression,followed by AzanOBP5 and AzanOBP4.In female adults,the expression of Aznan OBP4 in feet(P=0.000)and wings(P=0.018),AzanOBP5 in abdomen(P=0.001),feet(P=0.002)and wings(P=0.001),AzanOBP7 in wings(P= 0.001).In male adults,only the expression of gene AzanOBP7 in abdomen(P=0.000)was significantly higher than that in female.3.The prokaryotic expression vectors AzanOBPs/p ET-21 a of AzanOBP4,AzanOBP5 and AzanOBP7 were successfully constructed and the fusion protein His-AzanOBPs expressed in vitro was obtained.Two fusion proteins His-AzanOBP5 and His-AzanOBP7 were successfully purified and expressed in the supernatant.Western Blot verified that the purified fusion protein was the target protein.4.With the method of folding recognition,the three-dimensional models of protein AzanOBP4,AzanOBP5 and AzanOBP7 with fittable model quality were constructed with Amel OBP5,Agam OBP20 and Dmel GOBP1 as main templates.5.Semi-flexible docking was carried out with three A.zanthoxylum narrow-ginning proteins AzanOBP4,AzanOBP5 and AzanOBP7 as receptors and 46 host volatiles detected in the early stage of laboratory as ligands.Among them,the binding energy between AzanOBP4 and host volatiles was concentrated in-8.7 ～-3.9 k J/mol.The binding energy between AzanOBP5 and host volatiles ranged from-7.0 to-3.8 k J/mol.The binding energy of AzanOBP7 to host volatiles ranged from-7.1 k J/mol to-3.8 k J/mol.According to the overall binding energy,AzanOBP4 had better binding ability with host volatiles.Odor-binding protein was combined with host volatile receptor through hydrogen bonding,van der Waals force and hydrophobic interaction,but van der Waals force and hydrophobic interaction were more important.β-caryophyllene,humulene,trans-2-hexenal and trans-2-hexene-1-ol were screened out,which can be used as the formulation components of green chemicals for preventing and controlling A.zanthoxylum.The research results of odor binding protein of A.zanthoxylum were helpful to explore the molecular interaction mechanism between A.zanthoxylum and its host volatiles.Also study provided theoretical guidance for green prevention and control of A.zanthoxylum. |
