Binding Mechanism And Transcriptional Regulatory Of Odorant Binding Proteins Highly Expressed In The Antennae Of Monochamus Alternatus(Hope)

The sensitive olfactory sensing system plays an important role in the life process of insects,and odorant binding proteins(OBPs)are believed to participate in the first step of odor molecule recognition in the insect body,which is an important foundation for olfactory perception.Many studies have demonstrated that OBPs can bind to insect information chemicals and play an important role in insect olfactory responses;The expression specificity of OBPs in insects has been confirmed to be compatible with their physiological functions.However,due to the diversity of insects and their OBPs,the physiological functions of OBPs in insects are still not fully understood.In particular,transcriptional regulation is a key step in gene expression regulation,but it is currently unclear how Obps are regulated at the transcriptional level,which limits the exploration of the physiological functions of OBPs.Monochamus alternatus(Hope)is an important pest in forestry.Our laboratory identified several MaltObp genes through the transcriptome in the early stage,explored the function of some MaltOBPs,and initially found that three MaltObps(MaltObp3,MaltObp10 and MaltObp18)highly expressed olfactory function in the antenna of the longicorn beetle.In order to systematically analyze the physiological functions of the three MaltObps mentioned above in Monochamus alternatus,this study investigated the binding mechanism between MaltOBPs and ligands based on a systematic analysis of the spatiotemporal expression patterns of MaltObps,and explored the transcriptional regulation mechanism of MaltObps,in order to provide a theoretical basis for further exploring the physiological functions of MaltOBPs in Monochamus alternatus.The main research findings are as follows:1.Clarified the spatiotemporal expression pattern of MaltObps and the binding mechanism between MaltOBPs and ligandsThe MaltObp3,MaltObp10 and MaltObp18 were cloned and obtained(Gen Bank login numbers: KF460432.1,KF977563.1,KF977571.1,respectively).Analysis revealed that MaltObp3 and MaltObp10 belong to the Classic OBP family,with 6 conserved cysteine Cys sites;MaltObp18 belongs to the Minus-C OBP family and has four conserved cysteine Cys sites.The expression of the above three genes in different developmental stages of longicorn beetle was determined using q RT PCR.The results showed that MaltObp3,10 and 18 were expressed at high levels in the antennae of both male and female adults at various stages of development.The binding modes of MaltOBP3,MaltOBP10 and MaltOBP18 with binding active substances were analyzed by computer molecular docking technology.The results showed that the hydrophobic binding cavities of the three MaltOBPs mentioned above are mainly composed of non-polar amino acids,which bind to small molecule ligands through hydrophobic forces;The 101 st tyrosine Tyr in the MaltOBP3 binding cavity can form a hydrogen bonding force with(+)-limonene oxide,which is a potential key interaction site.2.Detected the transcriptional active region of MaltObp promotersWe cloned the complete genome sequences of MaltObp3,10 and 18 from Monochamus alternatus,clarifying the distribution of introns and exons in the gene.The MaltObp3 has a total length of 13936 bp,including 6 exons and 5 introns;The MaltObp10 has a total length of 5268 bp,including 7 exons and 6 introns;The MaltObp18 has a total length of 1260 bp,including 2 exons and 1 intron.Successfully cloned regulatory sequences of approximately 2000 bp upstream of the MaltObp3,10 and 18 initiation codons of Monochamus alternatus.Two positive regulatory active regions(-1622～-1523,-1318～-1215)and five negative regulatory active regions(-1720～-1622,-1418～-1318,-1215,-1115;There are three positive regulatory active regions(-1635～-1528,-313～-203,-100～-1)and five negative regulatory active regions(-1820～-1736,-1736～-1635,-1528～-1425,-416～-313,-203～-100)on the promoter of MaltObp10;There is one positive regulatory active region(-1800～-1600)and one negative regulatory active region(-1600～-1558)on the MaltObp18 promoter.3.Identification and analysis of the regulatory effect of transcription factors on MaltObp10Using the JASPAR insect transcription factor database,predict the potential regulatory elements in the negative regulatory region(-1736～-1635)and positive regulatory region(-100～-1)of the MaltObp10 promoter,and predict the potential action sites of two transcription factors(MaltCf2 and Maltonecut)in the negative regulatory region;The potential action sites of six transcription factors(MaltDr,MaltDll,Malthth,Maltnub,Maltrepo and Maltunpg)in the positive regulatory region.MaltDr,MaltDll,Malthth,Maltnub,Maltonecut,Maltrepo and Maltunpg belong to the Homeo domain factors family,while MaltCf2 belongs to the zinc finger gene family(C2H2 zinc finger factors).The transcriptional regulation activity of each transcription factor on the MaltObp10 promoter was determined by the dual-luciferase reporter experiment.The results indicate that transcription factors MaltDll,Maltrepo and Maltonecut can positively regulate the transcriptional activity of the MaltObp10 promoter;The transcription factors MaltCf2,MaltDr and Malthth have no significant regulatory effect on the transcriptional activity of the MaltObp10 promoter;The transcription factors Maltnub and Maltunpg can negatively regulate the transcriptional activity of the MaltObp10 promoter.Similarly,through the dualluciferase reporter experiment,we analyzed the transcriptional regulation activity of the transcription factors MaltDll and Maltrepo on MaltObp3 and MaltObp18 promoters.It was found that MaltDll and Maltrepo had a positive regulatory effect on the transcriptional activity of MaltObp3 and MaltObp18 promoters,or they were key transcription factors in the process of MaltObps gene transcriptional regulation.Using the JASPAR insect transcription factor database,predict the binding sites of transcription factors MaltDll,Maltrepo and Maltonecut on the MaltObp10 promoter.It was found that MaltDll has two binding sites in the transcriptional active region(-100～-1);Maltrepo has three binding sites in the transcriptional active region(-100～-1);Maltonecut has one binding site in the transcriptional active region(-1736～-1635).Through EMSA experiments,the in vitro binding of transcription factors(MaltDll,Maltrepo and Maltonecut)to predicted action sites was validated using transcription factor fusion proteins and biotin-labeled probes.It was found that both MaltDll and Maltrepo can directly bind to the predicted action site in the MaltObp10 promoter region,while Maltonecut cannot bind to this predicted auction site.In summary,this study identified the spatiotemporal expression patterns of three MaltObps highly expressed in the antennae of Monochamus alternatus,predicted the possible binding mechanism between MaltOBPs and ligands,and explored the direct binding of transcription factors MaltDll and Maltrepo to the MaltObp10 promoter region.The research results provide a theoretical basis for further exploring the physiological function of MaltOBPs in Monochamus alternatus.