Function Exploration Of The Odorant-binding Protein AhygOBP53 Gene In Agasicles Hygrophila

Alternanthera has developed into a global invasive plant due to its strong vitality and reproduction,and was listed as one of the first major invasive alien organisms in China.Agasicles hygrophila is used as a biological defence against insects because it feeds exclusively on A.philoxeroid.Insects’ sense of smell plays an important role in their development and odorant binding proteins（OBPs）are involved in the first step of the insect’s response to recognise molecules of environmental information compounds and can bind to a wide range of host plant volatiles.However,the role of OBPs in the recognition of environmental information compounds by the hymenopteran insect,the lotus grass,is not yet fully understood.In this paper,we found that the AhygOBP53 gene is highly expressed in the antennae of the lotus grass.In this paper,the evolution and olfactory functions of the AhygOBP53 gene were systematically investigated using techniques and methods such as gene cloning,phylogenetic analysis,spatio-temporal expression profiling,homology modelling,molecular docking,protein purification and fluorescence competitive binding assays,and the following conclusions were drawn:1.The full length of the AhygOBP53 gene was obtained by gene cloning,and the open reading frame（ORF）was found to be 384 bp,encoding 128 amino acids,with a molecular weight of 14.1 kD and an isoelectric point of 4.69.The amino acid sequence contains five conserved cysteine（Csy）residues and belongs to the Minus-C OBPs.Phylogenetic analysis showed that AhygOBP53 clustered with the coleopteran Colaphellus bowringi OBP23（CbowOBP23）in the same family,indicating that the two genes are most closely related.The spatio-temporal expression profile of the AhygOBP53 gene was analyzed using real-time fluorescence quantitative PCR.It was found that the expression of this gene was significantly higher in the adult stage than in other developmental stages,and the expression was significantly higher in female adults than in male adults,lower in the larval stage,and basically not expressed in eggs and pupae;in different tissue parts of the adult,the expression of the antennae was the highest,and the male antennae were significantly higher than the female antennae Therefore,it is hypothesized that AhygOBP53 plays an important role in the recognition of environmental information compounds by A.hygrophila.2.The 3D modeling of AhygOBP53 was constructed by using the homology modeling method with the online software Swiss-model,while the reliability of the model was evaluated using four algorithms provided by the online software SAVES,Procheck,Verify₃D,ERRAT and Whatcheck.The constructed AhygOBP53 model is mainly composed of six a-helices and irregularly coiled coils folded.The binding ability of AhygOBP53 to 98 A.philoxeroides odorant volatiles was studied by molecular docking technique,and the results showed that there were 26 compounds with binding energy less than-5.8 kJ/mol to AhygOBP53.This suggests that AhygOBP53 can bind to a wide range of host plant volatiles.3.A pure AhygOBP53 protein was obtained by prokaryotic expression and protein purification,and the binding of AhygOBP53 to 20 A.philoxeroid odor volatiles was detected using N-phenyl-I-naphthylamine（1-NPN）as a fluorescent probe,and the results showed that a total of 10 compounds with binding ability to AhygOBP53 were:phytane（Ki=58.49）,2,2,4-trimethyl-1,3-pentanediol diisobutyrate（Ki=50.93）,diethyl phthalate（Ki=39.40）,dextroterpine diene（Ki=31.37）,geraniol（Ki=25.75）,DMNT（Ki=23.68）,cypress brain（Ki=22.95）,phenylmethyl acetate（Ki=21.27）,leaf alcohol benzoate（Ki=19.94）,and（s）-cyclohexylethylamine（Ki=9.96）.The results of this experiment were in general agreement with the molecular docking results,indicating the reliability of the molecular docking technique for the rapid screening of ligands for AhygOBP53.This study illustrates the olfactory function of the AhygOBP53 gene of the biodefense insect A.hygrophila from the perspectives of bioinformatics and molecular biology,and clarifies the physicochemical properties,evolutionary status,expression and binding substances of AhygOBP53.The experimental results further enrich the studies related to the molecular mechanism of host specificity in A.hygrophila.