| Amitraz is a broad-spectrum formamidine pesticide which has good control effect on mites at all stages of development.It has been used for a long time to control harmful mites on fruit trees,vegetables,tea,cotton,soybean,sugar beet and other crops.Previous studies have found that amitraz acts on octopamine receptors of invertebrates through physiological experiments in vitro.There are various types of octopamine receptors in Tetranychus cinnabarinus（Boisduval）,and their downstream signaling pathways are also different,but there is no relevant research on which octopamine receptor is the main target of amitraz.Therefore,this paper selects an important agricultural mite Tetranychus cinnabarinus as the research object.First,the octopamine receptor genes were cloned and screened.By analyzing its molecular biological characteristics and pharmacological properties,the sensitivity detection of amitraz and the prediction of target binding sites were carried out,and the specific target of amphetamine in Tetranychus cinnabarinus was identified.The main results are as follows:1.The c DNA cloning and sequence analysis of Tc Octβ2R and Tc Tyr1R in T.cinnabarinusThe full-length c DNA sequences of octopamine receptor genes Tc Octβ2R and Tc Tyr1R were cloned through analyzing and screening T.cinnabarinus transcriptome database and T.urticae genome library.Via TMHMM server,it was found that the protein sequences encoded by the two genes have seven hydrophobic linearα-helix transmembrane structures belonging to typical G protein coupled receptor（guanine nucleotide-binding regulatory protein-coupled receptors,GPCRs）.Through multiple sequence alignment with octopamine receptors of other ticks,mites and insects,it was found that the two gene sequences had homologous conserved regions.By phylogenetic analysis with octopamine receptors of Coleoptera,Diptera,Lepidoptera,Hemiptera,Orthoptera and Hymenoptera insects and other species of Arachnida,the results showed that Tc Octβ2R and Tc Tyr1R were respectively clustered together with Octβ2Rs and Tyr1Rs of other species with high homology and close genetic relationship.Therefore,Tc Octβ2R and Tc Tyr1R respectively belong to type 2β-adrenergic-like octopamine receptor and type 1 octopamine/tyramine receptor.2.Bioassay and induced expression analysis of amitraz to T.cinnabarinusThe toxicity of amitraz to Tetranychus cinnabarinus was good,with LC50 value of25.02 mg/L.The changes of octopamine receptor gene transcription level after treatment of sublethal dose of amitraz were detected by real-time fluorescence quantitative PCR（Real Time Quantitative-Polymerase Chain Reaction,RT-q PCR）.The results showed that only Tc Octβ2R was significantly down-regulated,and the down-regulation response increased with the increase of amitraz concentration.It is speculated to be the target gene of amitraz.However,no significant differential expression of Tc Tyr1R was found between the treatment group and the control group,indicating that the gene is not sensitive to amitraz,it may not be the target gene of amitraz.3.Functional identification and sensitivity to amitraz of Tc Octβ2R and Tc Tyr1R in T.cinnabarinusThe recombinant expression vector of the target gene was constructed with pc DNA3.1（+）plasmid and expressed in mammalian cells.Octopamine and tyramine,the model ligands of octopamine receptor,could effectively activate Tc Octβ2R and Tc Tyr1R,and their activation ability was concentration dependent,indicating that Tc Octβ2R and Tc Tyr1R were functionally expressed in HEK-293 cells and CHO cells.Subsequently,the sensitivity of octopamine receptor to amitraz was tested.The sensibility of amitraz to Tc Octβ2R was about 24 times that of Tc Tyr1R(EC50:Tc Octβ2R(8.59×10-9M)>Tc Tyr1R(2.073×10-7M)).This indicates that amitraz can activate Tetranychus cinnabarinus type 2β-adrenergic-like octopamine receptor better than type 1 octopamine/tyramine receptor,proving that type 2β-adrenergic-like octopamine receptor Tc Octβ2R may be the main target gene of amitraz.4.Analysis of the binding sites of Tc Octβ2R and Tc Tyr1R with ligandsAccording to the predicted three-dimensional structure model,Tc Octβ2R and Tc Tyr1R have sevenα-helices,belonging to the typical GPCRs structure.Molecular docking results showed that Tc Octβ2R had better binding mode with amitraz than its mode ligand octopamine.But Tc Tyr1R had worse binding mode with amitraz than its mode ligand.Furthermore,the binding strength of amitraz and Tc Octβ2R was higher than that of Tc Tyr1R.Amitraz reacts with Tc Octβ2R through hydrogen bonding force at position Asp109.The free binding energy of three-dimensional structure model is-6.90Kcal/mol.Octopamine,the natural ligand of the octopamine receptor,also formed a hydrogen bond at Asp109,indicating there may be competitive binding between them.According to the results of multiple sequence alignment between T.cinnabarinus and other arthropods,it is found that the amino acid residues at position 109 of many species are aspartic acid（D）.It is speculated that Asp109 is the key site of amitraz acting on Tc Octβ2R.In conclusion,this research cloned and indentified two octopamine receptor genes of T.cinnabarinus,Tc Octβ2R and Tc Tyr1R.Furthermore,the toxic effect,target gene and specific binding site of amitraz on T.cinnabarinus were analyzed.It was proved that Tc Octβ2R is the target gene of amitraz to T.cinnabarinus,and Asp109 is the binding site.The findings will broaden the comprehension of physiological functions of octopamine receptors and construct a platform for the screening of agonists and antagonists.Furthermore,it is of great significance to develop new acaricides targeting octopamine receptors in T.cinnabarinus. |
