Functional Analysis Of Odorant Binding Proteins From Nilaparvata Lugens(ST(?)L)

Odor binding proteins(OBPs)in the antennae of insects play a significant part in insect olfaction and other physiological processes.The brown planthopper Nilaparvata lugens(Stal)is a serious migratory pest in rice producing countries in Asia.previous transcriptome sequencing of the antennae and other tissues of the BPH identified 10 NlugOBPs,which confirmed that NlugOBP3 is involved in olfactory activities in BPH and may have non-olfactory functions related to survival.However,the physiological function of other NlugOBPs remain unclear.Therefore,NlugOBP4,NlugOBP7,NlugOBP8 and NlugOBP9 genes were cloned and purified by prokaryotic expression system in vitro.The binding characteristics of NlugOBP8 and NlugOBP9 with 26 rice volatiles and their expression in Olfactory sensillum were investigated by fluorescence competitive binding,fluorescence quenching and circular dichroism(CD),homology modeling and molecular docking,H-type olfactometer and immunohistochemical localization.It is hoped to provide a basis for probing the physiological functions of NlugOBPs in the BPH.The main results are as follows:1.Cloning and sequence analysis of OBPs gene in the brown planthopperNlugOBP4(KC516720),NlugOBP7(KC663686),NlugOBP8(KC663687)and NlugOBP9(KC663688)coding sequences were cloned successfully,and the comparison with several OBPs sequences showed that:NlugOBP4,NlugOBP7 and NlugOBP8 have the characteristics of Classic OBP.NlugOBP9 belongs to Plus-C OBP.2.Spatial and temporal expression characteristics of OBPs in the brown planthopperQuantitative analysis of the spatial and temporal expression of the two genes at the transcriptional level was conducted by qPCR.The results showed that the expression levels of NlugOBP4 and NlugOBP7 were the highest in the first instar of BPH,and the gene expression levels decreased gradually with the growth of BPH.In the antennae of adult stage,both NlugOBP4 and NlugOBP7 had significantly highly expression,and the expression of NlugOBP4 among adults was independent of gender,while the expression of NlugOBP7 in female adults was obviously lower than that in male.3.Prokaryotic expression purification and binding characteristics analysis of NlugOBPs in brown planthopperProkaryotic expression and purification in vitro to obtain high-purity target proteins NlugOBP4,NlugOBP7,NlugOBP8 and NlugOBP9.The affinity of NlugOBP8 and NlugOBP9 with 26 rice volatiles was tested by fluorescence competitive binding assay.The results showed that under neutral conditions,NlugOBP8 could bind to 22 volatiles such as alkanes,terpenes,alcohols,aldehydes,ketones,esters,etc.Among them,NlugOBP8 had strong binding ability with 13 volatiles such as(-)-limonene,linalool and 2-tridecanone,while the other 9 volatiles had no binding ability.NlugOBP9 can bind to 6 volatile compounds,and the binding ability of NlugOBP9 is stronger than that of pH7.4 under the condition of pH5.0.Among them,the binding ability with methyl benzoate,benzaldehyde and cedrol was strong,and the binding ability with 3-carene,2-tridecanone and α-terpineol was weak.The quenching maps at different temperatures showed that among the 13 volatiles with strong binding ability,9 volatiles,such as nerolidol,linalool and p-cymene,were static quenched with NlugOBP8 and could form stable complexes.The other four volatiles were dynamically quenched with NlugOBP8.Static quenching occurred between methyl benzoate and benzaldehyde and NlugOBP9,while dynamic quenching occurred between cedrol and NlugOBP9.The results of CD showed that the main protein structure of NlugOBP8 was αhelicity and changed as the pH changes.In the pure protein solution,the content of αhelix was the highest at pH5.0,and the structure became more compact.The α-helix of protein solution also changes with different concentrations and different types of ligand.Homology modeling and molecular docking prediction results showed that the tertiary structure of NlugOBP8 protein had six α-helices,three disulfide bonds,and a hydrophobic binding cavity with a long C-terminal in the middle.The molecular ligands were bound by hydrogen bonds.The ligands were bound by hydrophobicity and hydrogen bonding at key sites such as Phe114 and Thr102.4.Immunolocalization of NlugOBPs in antennal sensilla of brown planthopperScanning electron microscope(SEM)was used to observe the morphological characteristics of the antennae of the brown planthopper,and the four main sensilla types and the special structure of chrysanthemum shaped sensory clusters were identified.The pure proteins NlugOBP8 and NlugOBP9 were obtained in vitro and the polyclonal antibodies anti-NlugOBP8 and anti-NlugOBP9 were immunized.The specificity of the two antibodies and the protein expression in different tissues were detected by western blot.In terms of protein levels,NlugOBP8 and NlugOBP9 were highly expressed in antennas,consistent with the expression characteristics of mRNA levels.Transmission electron microscopy(TEM)was used to observe the immunized antennae slices,and NlugOBP8 and NlugOBP9 were distributed in the trichoid,chaetica and auricillica sensilla of adults,which proved that NlugOBP8 and NlugOBP9 were likely involved in the process of BPH recognition of external odor molecules in the protein level.5.Screening of behavioral active substances by H-type olfactometerThe behavior responses of the BPH to rice volatiles initially screened by fluorescence competitive binding experiment and fluorescence quenching experiment were measured using H-type olfaction apparatus.The results showed that the repellent behavior of the BPH against trans-caryophyllene and linalool at 1‰ concentration disappeared after dsNlugOBP8 injection compared with dsGFP injection.When dsNlugOBP9 was injected,the significant tendency of BPH to benzaldehyde disappeared.The repellent behavior to methyl benzoate was significant.