| The green mirid bug(Apolygus lucorum)belongs to the order Hemiptera and family Miridae.It is a polyphagous pest widely distributed in cotton-growing areas of the Yangtze River and Yellow River basins in China.With the widely planting of Bt cotton,non-target pest outbreaks,especially the green mirid bugs,have caused serious economic losses to agricultural production.Green mirid bugs have a polyphagous and host-transferable habit,relying on their sensitive olfactory and taste systems to perceive complex external signals and respond accordingly.Currently,research on olfactory-related proteins in green mirid bugs mainly focuses on odorant-binding proteins(OBPs)and odorant receptors(ORs),while research on ionotropic receptors(IRs)is scarce.Based on the transcriptome sequencing and analysis of the green mirid bug,this study identified the IR genes of the green mirid bug and clarified the function of high-expressed IRs in the antennae,as well as analyzed their binding mode with ligands.1.Gene identification and expression analysis: Six IR genes were identified in the antennae of the green mirid bug: Aluc IR8 a,Aluc IR75 a,Aluc IR75 d,Aluc IR75 i,Aluc IR75 j and Aluc IR75 k.The complete open reading frame of Aluc IR8 a is 2562 bp,encoding 853 amino acids,while the sequence of the other genes varies from 1791 bp to 1902 bp.Pfam prediction results showed that Aluc IR8 a,Aluc IR75 a,and Aluc IR75 d contain at least one conserved ligand-gated ion channel functional domain(PF00060).Evolutionary analysis showed that Aluc IR8 a is highly conserved with other insect IR8 a.q PCR was used to investigate the expression patterns of these IRs in the antennae,head,foreleg,midleg,hindleg,and wings of males and females.The results showed that the six Aluc IRs were significantly higher expressed in the antennae than in other tissues.2.Functional analysis.Ligand screening of Aluc IR75 a,Aluc IR75 d,Aluc IR75 i,Aluc IR75 j and Aluc IR75 k was performed using Xenopus oocyte expression and two-electrode voltage clamp techniques.The results showed that the five co-expressed combinations of Aluc IR8 a with Aluc IR75 a,Aluc IR75 d,Aluc IR75 i,Aluc IR75 j,and Aluc IR75 k primarily responded to acidic amine compounds,with some main ligands being important plant volatiles.Furthermore,the electrophysiological responses of the green mirid bugs to the main ligands of the five IR complexes were measured by antennal recording techniques.The results showed that some main ligands could elicit electroantennography responses,and there was no significant difference between males and females.3.The binding mode of Aluc IRs with its ligands.The structures and key binding sites of Aluc IR75 a,Aluc IR75 d,Aluc IR75 i,Aluc IR75 j and Aluc IR75 k were analyzed using homology modeling and molecular docking techniques.Molecular docking experiments were conducted by modeling the structures and conducting tests with four ligands: nicotinic acid,salicylic acid,hexanoic acid,and trans-2-hexenoic acid in order to identify the key binding sites.The results showed that the amino acid residues TRP and ARG participate in the interaction between each gene and its corresponding ligands,speculating that they have important functions in the recognition of odor molecules in Aluc IRs genes.In summary,this study analyzed the function of six antennae-expressed IRs in green mirid bugs and proposed their involvement in the host-locating processes.These findings offer valuable references for investigating the function of IRs in green mirid bugs,facilitate a deeper understanding of the molecular mechanism of olfactory sensing in mirid bugs,and provide useful ideas for green pest control strategies against mirid bugs. |
PDF Full Text Request