草地贪夜蛾幼虫味觉受体SfruGR8和SfruGR9的基因克隆及配体结合特性分析OA

[Aim]The gustatory receptor genes SfruGR8 and SfruGR9,expressed in the larval head of the fall armyworm,Spodoptera frugiperda,were cloned and the ligand-binding properties of SfruGR8 and SfruGR9 were characterized,to lay the theoretical foundation for revealing the molecular mechanisms of sugar perception in S.frugiperda larvae.[Methods]Based on the transcriptome sequencing data of S.frugiperda larvae and the identification results of gustatory receptor genes,the cDNA sequences of SfruGR8 and SfruGR9 containing full-length open reading frames(ORFs)were cloned by PCR.The structural features of SfruGR8 and SfruGR9 were analyzed through phylogenetic analysis and multiple sequence alignment.Using the Xenopus laevis oocyte heterologous expression system combined with the two-electrode voltage clamp technique,the electrophysiological responses of the recombinant SfruGR8 and SfruGR9 to 11 candidate ligand compounds(inositol and 10 carbohydrates)were measured.The potential binding sites of SfruGR8 and SfruGR9 with glucose and fructose,respectively,were predicted via three-dimensional structural modeling and molecular docking simulations.[Results]The full-length cDNA sequences of SfruGR8(GenBank accession no.:LOC118262948)and SfruGR9(GenBank accession no.:LOC118274941)were cloned from S.frugiperda.SfruGR8 and SfruGR9 possess 8 and 7 transmembrane domains,respectively,characterized by a long intracellular N-terminus and a short extracellular C-terminus.SfruGR8 and SfruGR9 contain a conserved motif,-hh(G/A/S)(A/S)hhTYhhhhhQF,at the C-terminus and belong to the sugar receptor family.The recombinant SfruGR8 exhibited the greatest response to glucose,while the recombinant SfruGR9 showed an exclusive response to fructose.Both SfruGR8 and SfruGR9 showed dose-dependent responses to glucose and fructose,respectively.The recombinant SfruGR8 interacted with glucose via hydrogen bonds and van der Waals forces,with a binding energy of-25.26 kJ/mol,while the recombinant SfruGR9 bound to fructose through the same interactions,exhibiting a binding energy of-26.67 kJ/mol.[Conclusion]Both gustatory receptors SfruGR8 and SfruGR9 are sugar receptors.SfruGR8 shows a strong response to glucose,whereas SfruGR9 specifically binds to fructose.It is inferred that SfruGR8 and SfruGR9 play important roles in the feeding or digestion of sugar compounds in S.frugiperda larvae.