| The damage and insecticide resistance of stored-product insects have been serious problems for crop storage.Plant essential oils(EOs)have become a research hotspot in the control of stored-product insects because of their broad spectrum,safety,and low residue.Metabolic enzymes,such as the cytochrome P450(P450),glutathione S-transferase(GST)and esterase(EST)are involved in the degradation of plant EOs and their active constituents,but the mechanism is not clear.The slow efficacy and poor stability of EOs have become a major problem which seriously restricts the widespread application of EOs in agricultural production.The previous study showed that the essential oil of Melaleuca alternifolia and its main active ingredient terpinen-4-ol have significant fumigation activity against stored-product insects.Based on that,the synergistic effect of the P450 inhibitor-piperonyl butoxide(PBO)to terpinen-4-ol against Sitophilus zeamais was determined by a topical application method,and the role of P450 in regulating S.zeamais responding to terpinen-4-ol was evaluated.Based on RNA-seq and bioinformatics analysis,up-regulated P450 genes were screened to investigate their responses to terpinen-4-ol treatment,and the fxunction of target genes in the susceptibility of S.zeamais to terpinen-4-of was verified by RNAi technique.The results provide a theoretical basis for the study of the mechanism about stored-product insects in response to terpinen-4-ol,and provide a scientific evidence for the development of EOs as new environment-friendly insecticides for the control of stored-product insects.The main findings are as follows:1.Study on synergism of PBO to terpinen-4-ol against S.zeamaisThe PBO showed a synergistic effect to terpinen-4-ol against S.zeamais and the inhibitory effect on P450 enzyme activity in S.zeamais.When treated with 0.3 ?g of PBO,the mortality of tested insects responding to terpinen-4-ol fumigation increased significantly after 12 h.With synergist PBO treatment,the LC50 of terpinen-4-ol against S.zeamais decreased to 0.92 mg/L air,with a synergism ratio of 3.5-fold.Thus,the P450 enzyme system played an important role in S.zeamais responding to terpinen-4-ol.2.Transcriptional level analysis of S.zeamais in response to terpinen-4-ol and identification of major detoxification enzyme genesFunctional annotation and pathway analysis on the transcriptional level of S.zeamais in response to terpinen-4-ol fumigation were performed according to RNA-seq.The results showed that 36,117 unigenes were obtained after assembly.Aligned with Nr,Nt,Swissprot,KEGG,KOG,InterPro and GO databases,19,841,4,697,14,744,14,970,14,275,15,524 and 5,401 unigenes were successfully annotated,respectively.Based on the Nr annotation,54.79%of unigenes had the highest homology with Dendroctonus ponderosae Hopkins.Based on pathway analysis,592 differentially expressed genes(DEGs)were screened,in which detoxification-related DEGs included 16 P450 genes,8 GST genes,14 EST genes,10 UDP-glucuronosyltransferase(UGT)genes and 2 ATP-binding cassette transporter genes,with the log2 ratio ranged from-2.57 to-1 and 1 to 4.21.Among them,P450 genes had higher up-regulation,indicating P450 played an important role in the metabolism of terpinen-4-ol in S.zeamais.3.Response pattern analysis about P450 genes of S.zeamais induced by terpinen-4-olBased on the results of the transcriptome analysis,RT-qPCR was used to detect the expression pattern of 16 P450 genes in response to terpinen-4-ol treatment.The results showed that expression levels of P450s were significantly altered under different concentrations(LC20,LC50 and LC80)of terpinen-4-ol.When treated with LC50 of terpinen-4-ol,the expression levels of P450s increased significantly at different time.The dose and time effects of P450s in response to terpinen-4-ol were significant.The up-regulation of Unigene5887_All and CL451 l.Contig3_All were significant,which were evaluated to be the key genes in the detoxification of terpinen-4-ol in S.zeamais.4.Bioinformatics analysis of P450 reductase gene and P450 genes related to terpinen-4-ol stressThe full-length cDNA sequences of Unigene5887_All and CL4511.Contig3_All were obtained by cloning,which were named as CYP4DV2 and CYP6MS1,respectively.Cytochrome P450 reductase gene was named as SzCPR.Bioinformatics analysis showed that SzCPR had two transcripts SzCPR-X1 and SzCPR-X2,both of which have four functional domains:member anchor,FMN binding,FAD binding and NADP binding site.CYP4DV2 and CYP6MS1 belonged to the CYP4 family and the CYP6 family,respectively.Furthermore,CYP4DV2 and CYP6MS1 had characteristics of P450 super family and belonged to the microsomal cytochrome P450 genes.5.Study on the role of SzPCR and P450 genes in the susceptibility of S.zeamais to terpinen-4-ol using RNAiThe RNAi system of the target genes in S.zeamiais was established successfully.The results showed that the silence of SzCPR and CYP6MS1 genes increased the susceptibility of S.zeamais to terpinen-4-ol,with the mortality increased 25%and 27%under the treatment of LC30 of terpinen-4-ol,respectively(p<0.05).However,knockdown of CYP4DV2 increased the mortality of S.zeamais by 10%,with no significant difference compared to controls.The results indicated that SzCPR and CYP6MS1 mediated the susceptibility of S.zeamais to terpinen-4-ol and could play an important role in the detoxification and metabolism of terpinen-4-ol in S.zeamais. |
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