Identification And Function Verification Of Two Monoterpenes Metabolized Genes In Maize Weevil,Sitophilus Zeamais Motschulsky (Coleoptera:Curculionidae)

The maize weevil（Sitophilus zeamais Motschulsky）is the most destructive stored- product insect in China,it is also the one of most important stored-product insects around the world which is one of the boring insects.The damage of stored-product pest is always the most important factor of the food storage and quality.Since the superior chemical fumigation reagent bromomethane was banned in the whole world,the resistance of stored-product insects to phosphine was increasing and the formation of sulfuryl fluoride residues as a potent greenhouse gas has caused widespread concern.The a few fumigations such as phosphine are facing the challenage.Limit of conservative cheimical pesticide control has been occurred in the field of pest resistance,environment pollution and human security.Biofumigation was concerned the effcient pathway of control stored-product pest.In the previous study,we found 2 enesstial oils from Melaleuca alternifolia and Taxodium zhongshansa shown outstanding fumigation activity against to S.zeamais,and the main activity component was terpinen-4-ol and limonene by gas chromatograph-mass spectrometer.However,the way of detoxification metabolism of S.zeamais to both reagent has still unkown.In this graduation thesis,RNAi was used to tentatively explore the detoxification metabolism function of P450 genes which response to terpinen-4-ol and limonene in S.zeamais,then to simulation study the docking site between P450 and terpinen-4-ol and limonene using molecular docking.The results were as follows.1.Biotransformation of terpinen-4-ol and limonene by S.zeamaisThe metabolites of maize weevil treated with terpinen-4-ol and limonene for 24 h were detected by GC-MS.There are pathways of terpinen-4-ol and limonene in maize weevil,and the metabolite of terpinen-4-ol may be p-menth-1-en-1,4-diol;the metabolite of limonene may be 1,3,8-p-menthatriene.2.Clonging and sequence analysis of P450 genes in S.zeamaisFive p450 genes related to terpinen-4-ol and limonene were identified through the analysis of the transcriptome data of S.zeamais.The full-length c DNA sequences of the five P450 genes were cloned and verified by RT-PCR,and these genes were named as CYP6MS1,CYP6MS5,CYP6MS6,CYP6MS8 and CYP6MS9 by Dr.David Nelson.And thses genes encodes 494,493,494,494 and 493 amino acids,respectively.After amino acid multiple sequence alignment,the results showed that the sequence identity of the five P450 genes was more than 80%,and has all complete 5 motifs,namely WxxLR（helix C）,GxxT（helix I）,ETLR（helix K）,PE/DKFNPENF（P450 meander）and FGD/EGPRMCIG（heme binding loop）,except for helix C and I,the other three motifs show quite conserved.3.Expression patterns of CYP6MS genes in S.zeamaisBy qPCR,the expression patterns of maize weevil P450 gene under different drug and concentration stress,and at different developmental stages,different parts and tissues were quantitatively detected.The results showed that except CYP6MS9,the expression levels of CYP6MS1,CYP6MS5,CYP6MS6 and CYP6MS8 were significantly induced by terpinen-4-ol and limonene.And at different developmental stages,CYP6MS1 was highly expressed in1st and 2nd instar larvae and adults;CYP6MS5 was highly expressed in 1st instar and pupal stage;CYP6MS6 and CYP6MS8 were highly expressed in all instars except pre-pupa,and expression level CYP6MS9 was highly expressed in adults.In different parts and tissues,except CYP6MS5,the other four genes have the highest expression levels in wings,while CYP6MS5 has the highest expression levels in thorax.4.Terpinen-4-ol and limonene fumigation activity changes after RNAiThe CYP6MS gene was silenced by feeding maize weevil adults with bacterially expressed dsRNA,and the results showed that the expression of each gene could be effectively knocked down,up to 98%compared with the dsEGFP feeding control;Under terpinen-4-ol fumigation,the mortality rate increased by 25%,25%,16%,and 17%,respectively;under limonene fumigation,the mortality rate increased by 29%,25%,15%,and 22%,respectively after feeding dsCYP6MS1,dsCYP6MS5,dsCYP6MS6 and dsCYP6MS8.However,there was no significant difference in the feeding dsCYP6MS9experiment.The results indicate that CYP6MS gene knocked down can caused the increased sensitivityo 4-terpineol and limonene in S.zeamais.5.Molecular docking of CYP6MS with Terpinen-4-ol and limoneneThe three-dimensional structure of CYP6MS protein was predicted by Alphafold2,and the molecular docking was performed using Discovery Studio 2021 software.The results showed that both terpinen-4-ol and limonene could dock with five CYP6MS proteins.The main binding mode of CYP6MS and terpinen-4-ol is through hydrogen bonding,van der Waals force and hydrophobic interaction;it is mainly combined with limonene through van der Waals force and hydrophobic interaction,but the binding mode is mainly van der Waals force and hydrophobic interaction.Through binding energy analysis,it was found that the binding energies of terpinen-4-ol and CYP6MS ranged from-15.6333 to-42.328 kJ·mol^-1,and the binding energies of limonene and CYP6MS ranged from-10.4564 to-36.5125 kJ·mol^-1.On the whole,terpinen-4-ol and limonene had better binding ability to S.zeamais CYP6MS,terpinen-4-ol and limonene are combined with P450 receptors through hydrogen bonding,van der Waals force and hydrophobic interaction,but van der Waals force and hydrophobic interaction are more important.The above results show that the CYP6MS subfamily genes are related to the detoxification function of terpinen-4-ol and limonene in S.zeamais.This provides theoretical support for the development and application of RNAi pesticides and also lays a foundation for the development of plant essential oil fumigants.