| The prevention and control of stored grain pests is the key work to ensure the safety of grain reserves.Plant-based repellents have a series of advantages,such as not easy to make pests resistant and friendly to the environment,and they have become potential substitutes for synthetic repellents.Odorant receptors(ORs)are the most critical links in the process of insect olfactory system sensing repellents.In this paper,we used limonene,a plant-derived peticidies active ingredient,as an experimental drug to clarify the repellent activity of limonene against adults Tribolium confusum and the electroantennogram(EAG)response of T.confusum through repellent test and electroantennogram.Moreover,the functions of the potential target ORs genes involved in the process of identifying limonene in the antennae of T.confusum were screened and verified by transcriptomics,fluorescence quantitative PCR and RNA interference(RNAi).The main results are as follows:1.Determination of repellent activity and EAG response of limonene to adults of T.confusumThe repellent activity was measured by the filter paper residual film method.The test results showed that with the increase of limonene concentration and the decrease of repellent time,the repellent rate against the adults of T.confusum gradually increased,and when the limonene concentration was 200μg/cm~2,and its repellent level remains level V at 24 h.EAG bioassay results showed that the maximum EAG response value caused by limonene was 0.90±0.81 m V for female and 0.59±0.50 m V for male.With the increase of concentration,the EAG signal of both males and females increased,indicating that limonene can be recognized by the antennae of T.confusum and activate its olfactory sensory neurons.2.Analysis of antennal transcriptome sequencing and screening of related olfactory genes for limonene perception by T.confusumThrough the transcriptome sequencing of the antennae of T.confusum in the limonene treatment group and the blank control group,70977 unigenes were obtained by assembly and splicing,with an average length of 1964 bp,and 54067 unigenes were successfully annotated.Based on the transcriptome data,186 differentially expressed genes were found,of which 88 genes increased and 98 genes decreased.According to the functional annotation and BLAST comparative analysis of the differential genes,11genes related to smell were screened,including 7 ORs genes,1 SNMPs gene and 3genes involved in coding ODEs genes.With the log2 ratio ranged from-6.78 to 4.33.The results of transcriptome sequencing were verified by q RT PCR.It was found that the expression level of four ORs genes including Unigene4817＿All、CL762.Contig2＿All、CL3391.Contig4＿All、CL1796.Contig11＿All decreased significantly and the expression level of one ORs gene CL3229.Contig5＿All increased significantly,indicating that the five ORs genes may play an important role in the process of identifying limonene odorant molecules in the antennae of T.confusum.3.Cloning and sequence analysis of five Tcon ORs genesFive ORs gene sequences were cloned and named Tcon OR93,Tcon OR94a,Tcon OR22c-like,Tcon OR139 and Tcon OR1 respectively.The open reading frames contained in the whole length encode 387,398,377,182 and 376 amino acids respectively.They all have a conserved domain 7tm-6 superfamily of insect ORs family,which is consistent with the structural characteristics of insect traditional ORs.The phylogenetic tree showed that the five ORs genes were closely related to the ORs of Tribolium castaneum.4.Study on the function of five ORs genes in T.confusumThrough RNAi technology,combined with repellent test and EAG response,the function of ORs gene for recognizing limonene in adults of T.confusum was verified.By microinjection method,we introduced ds RNAs of five ORs genes into the pupae of T.confusum.After 72 hours,it was detected that the silencing efficiency of the five ORs genes was 53.84%-85.07%.When the gene silenced test insect matures,the results showed that after silencing Tcon OR94a,Tcon OR22c-like and Tcon OR1 genes,the repellent and EAG responses to limonene did not change significantly.The repellent effect and EAG response value of limonene on T.confusum injected with ds Tcon OR93and ds Tcon OR139 decreased.However,Tcon OR93 was the most significant different from the control group,indicating that Tcon OR93 may be more involved in the process of limonene repellent against the adult of T.confusum. |
