Binding Characteristics Of Four Antennal-specific Odorant Binding Proteins In Apolygus Lucorum(Hemiptera:Miridae)

The large plantation of transgenic BT cotton in our country improves the governance for cotton field significantly,reduced the usage of pesticides and controlled the cotton bollworm effectively,but the number of the green bug(Apolygus lucorum)secondary pests of cotton filed increased dramatically,damage lots of economic plants heavily.The green bug(A.lucorum)olfactory system play an important role in the host location,mating and avoid predation.Study intensive on the olfactory system have profound guiding significance for designing behavior regulator.Odorant binding proteins(OBPs)play a key role in the olfactory system,further study the odor recognition spectrum of OBPs would promote the analysis of olfactory system and provide new ideas for the prevention and control of the green bug.Prophase research of this laboratory have identified 38 OBPs in the green bug.In this paper,ligand binding characteristics of four antennal-specific expressed Aluc OBPs(Aluc OBP14?Aluc OBP21?Aluc OBP22?Aluc OBP24)were under reviewed.The main contents are as follows:1.Cloning,expression and purification of four antennal-specific expressed Aluc OBPs,four recombinant proteins of Aluc OBPs were obtained.Study the binding characteristics of these four recombinant proteins though fluorescence competitive binding assay with 68 chemicals,68 candidate ligands including latent sex pheromone and sex pheromone analogs,terpene compounds and green leaf volatiles.Results show that Aluc OBP14 can bind with beta-ionone,lauraldehyde,hexyl hexanoate,octyl butyrate;Aluc OBP21 can bind with beta-ionone,beta-caryophyllene;Aluc OBP22 can bind with beta-ionone,beta-caryophyllene,hexyl hexanoate and octyl butyrate;Aluc OBP24 can bind with beta-ionone,beta-pinene.It indicate that four OBPs may perform an important role in olfactory system.Especially,Aluc OBP14 and Aluc OBP22 can bind with hexyl hexanoate,a sex pheromone analog,it infer that these two OBPs can both function as PBP and GOBP.All OBPs can bind with beta-ionone,it hypothesized that beta-ionone was an important ligand for Aluc OBPs.Moreover,parallel function study of Aluc OBP21 used Micro Scale Thermophoresis(MST)showed that the MST result basically consistent with luorescence competitive binding assay,but binding spectrum of MST was broader with a smaller and preciser dissociation constant.2.Analysis of the structure and key functional sites of Aluc OBP22 use Homology modeling and Molecular docking.Construct 3D structure of Aluc OB22 template as Mvic OBP3(4Z39),then dock with beta-ionone,beta-caryophyllene,hexyl hexanoate and octyl butyrate,searching for critical binding sites.Results show that 3Asp,5Leu,40 Ile,41Met,44 Val and 45 Met may perform a key role in binding with odors.Site directed mutagenesis experiment test found that after 5Leu and 44 Val mutant to ALA the mutantion lose the binding ability to beta-caryophyllene,hexyl hexanoate and octyl butyrate,but remain binding ability to beta-ionone,it may result from the special construction of beta-ionone.The above test show that 5Leu and 44 Val were the key sites of Aluc OBP22.