Functional Analysis Of Neuropeptide F And Its Receptor Genes In Hyphantria Cunea(Drury)Based On RNAi Technology

Hyphantria cunea is a worldwide quarantine pest which causes severe damage to different plants resultiing in social economic loss and ecological environment loss.Insect NPF can activate G protein coupled receptors to regulate a variety of physiological processes and destroy signal transduction of insect neuropeptide in order to control pest.In this study,the entire length of the NPF/NPFR gene cDNA was cloned from Hyphantria cunea,and RNAi technology(dsRNA and siRNA)was used to study the function of the silenced gene.The function of NPF/NPFR gene and the function analysis of NPF/NPFR gene on feeding,reproduction and starvation stress of Hyphantria cunea.The specific conclusions are as follows.1.HcNPF,HscNPF,HcNPFR,HcsNPFRA10 and HcsNPFRA11 full-length gene sequences were successfully identified.The gene reading frame(ORF)is 246 to 1401 bp in length and encodes 81 to 466 amino acids.The molecular weight of the protein is 9.25kDa to 52.94kDa.PI is 6.73?9.25.HcsNPFRA10 is a acidic protein and the rest are basic proteins.Both HcNPF and HscNPF contain signal Peptides,The canonical amidation site(G)was found at the C-terminus of the mature peptide.The C-terminus of HcNPF protein is RPRF amide and the C-terminus of HcsNPF protein is RLRF amide.HcNPFR,HcsNPFRA10 and HcsNPFRA11 do not contain signal peptide sequences and belong to the GPCRs A family.2.RT-qPCR method was used to analyze the development and tissue expression specificity of NPF and NPFR genes.The results show that:HcNPF and HcNPFR genes were expressed in each developmental stage of Hyphantria cunea.The expression levels varied alternately with the growth of the instar in the larval stage and were higher in the adult stage.The expression level is high.and the expression levels of HcsNPF and HcsNPFR genes at certain developmental stages show certain synchronization.And There is a higher expression in the midgut and testis and ovary.This specific expression pattern indicates that NPF and NPFR genes are involved in regulating the feeding and reproductive functions of Hyphantria cunea.3.Use RNAi technology to microinject HcNPF and HcNPFR gene-specific dsRNA into the 4th larvae of Hyphantria cunea to obtain gene silencing of Hyphantria cunea.RT-qPCR was used to detect the transcription levels of the corresponding target genes at 24h,48h,72h and 96h time points after injection of dsRNAHcNPF and dsRNAHcNPFR.The research results show that HcNPF and HcNPFR genes can be effectively silenced by microinjection,and the 96h silencing effect is the best.The silencing efficiency is 32.71%and 88.64%,respectively.Compared with the control group,the feed intake of larvae injected with HcNPF and HcNPFR gene dsRNA was decreased by 31.75%and 17.55%,and the cumulative growth rate of body weight was decreased by 31.89%%and 13.54%,respectively.These results show that the HcNPF and HcNPFR gene silencing have a significant effect on the larvae's feed intake and cumulative growth rate of body weight.The target gene silenced larvae were given starvation stress.After 96h of starvation,the survival rate of the control group was significantly lower than that of the HcNPF and HcNPFR gene interference groups.These results indicate that the HcNPF and HcNPFR genes have positive regulatory effects on the starvation resistance of Hyphantria cunea.HcNPF and HcNPFR gene silenced adult emerged and mate,the female adult in the treatment group had significantly lower egg production than the control group.The number of spawning decreased by 70.86%and 59.87%,respectively.This indicates that Hyphantria cunea pupae after injection of dsRNA of HcNPF and HcNPFR genes inhibited the oviposition number of mated adults after mating,indicating that the HcNPF and HcNPFR genes may be involved in regulating the reproductive function of Hyphantria cunea.4.The siRNA-mediated RNAi silencing of HcsNPF and HcsNPFR genes in Hyphantria cunea larvae was performed by microinjection.The results show that HcsNPFR gene can be effectively silenced by microinjection.After the silencing of the HcsNPFR gene in 4th instar larvae for 72h,the silencing effect of the targets HcsNPFRA10-1 and HcsNPFRA10-2 was the best at 71.44%and 81.15%,respectively.Compared with the control group,the feeding intake decreased by 81.26%,83.31%.And the weight growth rate decreased by 39.08%,32.92%.The target HcsNPFRA10-3 had the best silencing effect at 48h(53.61%),when the feeding intake decreased by 71.24%and the growth rate of body weight decreased by 11.94%.The three targets of HcsNPFR-A11 gene all had the best silencing effect at 72h,which were 83.10%,90.81%and 80.97%,respectively.The feeding intake decreased by 72.51%on average and the weight growth rate decreased by 40.50%on average.After silencing the HcsNPFR gene in 5th instar larvae,among all the siRNA targets of HcsNPFR-A10 gene,the target HcsNPFRA 10-2 showed the best silencing effect at 48h(83.60%).Compared with the control group,the feeding intake decreased by 36.30%and the growth rate of body weight decreased by 46.92%.Among all the siRNA targets of HcsNPFR-A11 gene,the target HcsNPFRA11-2 had the best silencing effect at 72h(87.00%).The feeding intake decreased by 58.90%and the weight growth rate decreased by 49.40%.After silencing the HcsNPFR gene,the larvae's feed intake will be significantly reduced,and the cumulative growth rate of body weight will be significantly reduced.Interfering with the HcsNPFR gene will inhibit the feeding and weight gain of Hyphantria cunea,and then affect the normal growth and development of Hyphantria cunea.