| The chemical information communication between insects and environment play a vital role in insects. For example, insects locate host plant through volatiles; mating behaviour of insects relies on the pheromone or other chemicals. In the control of pest, we can development new and effective biopesticide based on the research of olfactory recepting mechanism which has the practial significance. Odorant-binding protein (OBP) is an important protein in the olfactory recepting, and the definite function of this protein is not clear. The brown planthopper (BPH, Nilaparvata lugens (Stal)) is a specialist herbivore and migrant pest on (wild) rice. The BPH adults and nymphs like feeding abundanting on the dark and wet rice base. With the resistance of BPH going up, it’s hard to control the damage using chemical control method. Here, we studied odorant-binding proteins (OBPs) of the BPH, base on genes cloning, we analyzed the expression pattern of the OBP genes and the binding properties with the odorant. In order to do a tentative exploration on the function of OBPs, we laid the foundation for the development of the prevention and control technology for BPH. The major results of this paper are list as below:1Cloning and sequence analysis of OBPs of BPHIn the paper, four OBP genes were cloned, NlugOBP4, NlugOBP5, NlugOBP6, NlugOBP7, and all of them possess complete open reading frame. The length is514bp,495bp,591bp,426bp respectively, coding167,164,196,141amino acids respectively. The ID in GenBank is KC663683, KC663684, KC663685, KC663686separately. The blast of four sequences show the all possess six conserved cysteine sites, no site lose or plus, meaning the four OBPs belong to Classic OBPs. And the prediction of signal peptide analysis of N-terminal show NlugOBP6and NlugOBP7contain19and20amino acids respectively, while the other two do not have.2Temporal and spatial expression profiles of NlugOBPs genesUsing real-time PCR detect the expression of the NlugOBP6, NlugOBP7, NlugOBP8, NlugOBP9, show the four genes all mainly expression in the antenna of BPH. With the development of BPH, the expression of the four genes present different trend. Overall, in a single time, the expression level of genes in female are higher than in male. There is no significant difference between long-wing and short-wing, except for some time. In short-wing female BPH, the expression levels of genes are impacted obviously by mating behavior. At3days, the expression levels of four genes in unmated females are higher than in mated females (P<0.05). In mated5-day-old long-wing BPHs, the expression levels of NlugOBP6are higher than in unmated (P<0.05). In contrast, NlugOBP6in unmated is higher than in mated short-wing5-day-old males (P<0.01). We speculated the expression levels of these genes are related to the behavior of the BPH with the analysis of the expression profiles of NlugOBPs genes.3The prokaryotic expression, purification and fluorescence competitive binding assays of NlugOBPsIn our research, the NlugOBP4is successfully constructed into pET-22b. The recombinant plasmid is expressed in E.coli BL21(DE3). The target protein mainly present as inclusion bodies. Extracts were purified with DE52and QFF affinity column, after isolation, dissolution and refolding.In neutral (pH=7.4) and acid (pH=5.0) environment, fluorescence competitive binding assay was used to test the binding characteristics of NlugOBP to37kinds of rice volatiles respectively, used1-NPN as a probe. The binding abilities of the NlugOBP4in neutral are better than in acid. In neutral environment, NlugOBP4show better binding abilities with Ketones, Aldehydes and most Terpenes, some Alkanes and Alcohols come later. The results of the test are consistent with the behaviour test previously reported before. Maybe these volatiles can be used as the main composition of the attractant or biopesticide of BPH. |
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