Identification And Functional Characterization Of The Neuropeptide F Receptor In Plutella Xylostella

Plutella xylostella is a lepidopterous pest that mainly affects cruciferous vegetables and is resistant to a variety of insecticides currently applied for control.The study of control strategies based on novel mechanisms of action becomes particularly important.Neuropeptides and their receptors are widely distributed and highly conserved in insects and are involved in regulating numerous physiological behaviors and processes in insects,which are considered as important potential biological targets for pest control.Neuropeptide F(NPF)and its receptor(NPFR)were found to be involved in regulating physiological functions such as feeding,reproduction,learning,and locomotion in a variety of insects,but less research has been done on NPF and NPFR in P.xylostella.In this study,the NPF and NPFR genes of were cloned and bioinformatically analyzed,and the spatiotemporal expression characteristics of NPFR were investigated.The effects of NPFR on the growth and feeding of larvae were investigated by RNA interference(RNAi)technology,and the effects of starvation and toxin stress on the expression level of NPFR were also studied.The prokaryotic expression system of NPF and eukaryotic expression system of NPFR were successfully constructed,and the binding characteristics and interactions between ligands and receptors were analyzed by biolayer interferometry and molecular docking,which provide reference for the development of new pest control methods using NPFR as the target.The main findings are as follows:(1)NPF-1,NPF-2 and NPFR were successfully cloned from P.xylostella and bioinformatics analyzed.The coding sequences of Px NPF-1 and Px NPF-2are 372 bp and 249 bp,encoding 123 and 83 amino acids,with predicted prot ein molecular weights of 14.14 k Da and 9.53 k Da,all of which are unstable a lkaline hydrophilic proteins.The first 24 amino acid sequences were are signal peptide sequences,which are secreted proteins.The coding sequence of Px NP FR is 1149 bp,encoding 382 amino acids.The predicted molecular weight of the protein is 42.8 k Da.It is a stable alkaline hydrophilic protein.The protein has seven α-helix transmembrane domains and belongs to GPCR superfamily.(2)The expression patterns of Px NPFR gene in different developmental stages and tissues were determined by q RT-PCR.Px NPFR was expressed in different instars and tissues,and the highest expression level was found in the 4th instar and midgut,and the lowest expression was found in adults,indicating that Px NPFR gene may be related to the feeding behavior of P.xylostella larvae.(3)The effects of silencing NPFR gene on the growth and feeding of P.xylostella were studied by introducing specific ds RNA by feeding.The results showed that the expression of the target gene NPFR could be effectively reduced by feeding ds RNA.After continuous silencing of NPFR gene,the larvae in the experimental group had delayed development,smaller morphology and significantly lower body weight than the control group(p<0.05).After feeding ds RNA for 12 h,the larvae were put into normal feed to develop until pupation,and the feeding quantity was higher than that of the control group and the pupation time was delayed for about 24 h.Silencing of the NPFR gene inhibits body weight gain,delays development,delays pupation and affects growth and development by regulating the feeding behavior.(4)Both toxin stress and starvation stress caused significant down-regulation of the target gene Px NPFR(p<0.01).It is hypothesized that the down-regulation of NPFR gene expression level correlates with the inhibition of feeding after feeding on Cry1 Ac toxin by the larvae of P.xylostella.NPFR was depleted under starvation stress,further suggesting that NPFR is involved in the regulation of feeding behavior.(5)The results of biolayer interferometry and molecular docking analysis based on Alphafold2 modeling showed that the endogenous ligands NPF-1 and NPF-2 were strongly bound to NPFR.NPF-1 and NPF-2 mainly form interactions with helices I,II,III,and IV of NPFR,and mainly involved in binding by hydrogen bonding and hydrophobic interactions.The key binding sites for NPF-1 and NPFR interactions were Tyr64,Tyr158 and Arg247,and the key binding sites for NPF-2 and NPFR interactions were Tyr22 and Tyr188,respectively.