Functional And Transcriptional Regulation Analysis Of MaltObp9 In Monochamus Alternatus

The olfactory system plays a key role in insect life activities,and olfactory proteins are the basis for the function of the olfactory system.Among them,odorant binding proteins(OBPs)are a class of olfactory proteins distributed in the lymph of insects,and are believed to function as carriers of hydrophobic compounds in the lymph.Under the induction of information chemicals,the expression of insect Obps gene changes,which in turn mediates the corresponding behavior of insects.However,it is not clear how the Obps gene is induced and regulated.Gene transcription regulation is an important part of gene expression regulation mechanism.However,only a few Obps transcription regulation and transcription factors have been reported so far.Due to the diversity of OBPs species in insects,it is still an important direction to explore the key transcription factors involved in the regulation of Obps gene expression induced changes.Monochamus alternatus Hope is an important vector insect of Bursaphelenchus xylophilus,a major forestry quarantine disease in our country.The research group’s previous research found that MaltObp9 is distributed in various sensilla of its antennae,and can bind to a variety of pine volatiles,which has potential olfactory function.However,the binding mechanism of MaltOBP9 and volatiles and the regulation mechanism of MaltObp9 are still unclear.Therefore,based on the analysis of MaltOBP9 expression characteristics and secretion activity,the binding characteristics and mechanism of MaltOBP9 and volatiles were analyzed,and the molecular mechanism of MaltObp9 gene transcriptional regulation was explored.In order to provide theoretical basis for exploring the function of MaltObp9 in olfactory perception of Monochamus alternatus Hope and its transcriptional regulation mechanism.The main results are as follows:The expression characteristics of MaltObp9 and secretion characteristics of MaltOBP9were clarified.The expression of MaltObp9 in olfactory organs of male and female adults of Monochamus alternatus Hope at different ages was detected by q RT-PCR.The results showed that MaltObp9 could be highly expressed in antennae and maxillary palpus of male and female adults at different days of age.The CDS sequence of MaltObp9 gene was cloned.MaltOBP9 contained 19 amino acid signal peptide sequences.The secretory activity of MaltOBP9 signal peptide was analyzed by TTC staining,and the results showed that the signal peptide of MaltOBP9 had secretory activity.The secretion of MaltOBP9 was verified using HEK293T cell line.The results showed that MaltOBP9 could be secreted extracellular.Thus,MaltOBP9 has secretory capacity and can be synthesized in the cell and then secreted into the extracellular environment to perform its function.The binding properties of recombinant MaltOBP9 to ligands were discovered.Based on the prokaryotic expression of soluble pure protein,the binding characteristics between recombinant MaltOBP9 protein and 22 volatiles were detected by fluorescence competitive binding experiment.The results showed that p H had a significant effect on the binding of recombinant MaltOBP9 to the ligand.In a neutral environment(p H=7.4),18 of the 22tested volatiles could bind to MaltOBP9(K_i<50μmol/L),and 13 of them had a strong binding ability to MaltOBP9(K_i<20μmol/L),indicating that MaltOBP9 can identify various types of volatiles in sensory lymph in a neutral environment,and has a broad binding spectrum.In an acidic environment(p H=5.0),all ligands except Longifolene had a weaker binding capacity to MaltOBP9 than the neutral condition.The binding mechanism of MaltOBP9 and ligand was analyzed,and the key sites of MaltOBP9 binding to ligand was clarified.The 3D structure of MaltOBP9 was obtained by ab initio modeling.Analysis of the structure showed that MaltOBP9 has a compact structure,containing 7α-helices to form a relatively closed binding cavity,and most of the binding cavity belongs to the hydrophobic region.MaltOBP9 contains four conserved cysteines(Cys),which are consistent with the characteristics of Minus-C OBPs family and form two disulfide bonds.Through the molecular docking of MaltOBP9 and 18 kinds of volatiles with binding ability,it was found that all the tested volatiles could bind in the binding cavity of the protein.The amino acid residues Phe54 and Glu104 in the binding cavity can form hydrogen bonds andπ-πinteractions to enhance the binding ability with proteins.Through the site-directed mutation of Phe54 and Glu104,it was found that Glu104 played a key role in the process of MaltOBP9 binding the pheromone 2-Methoxy-4-vinylphenol,and Phe54participated in the binding of all volatile molecules in the hydrophobic cavity,and the protein lost its binding ability with all volatiles after the mutation Phe54,and Phe54 was considered to be a key site for MaltOBP9 binding volatiles.Transcription factors that may be involved in the transcriptional regulation of MaltObp9 were screened out.The MaltObp9 gene CDS upstream sequence of 1668 bp was obtained by cloning.The MaltObp9 locus was predicted by the online website and analyzed by the genomic data.The MaltObp9 gene has a total of 1794 bp,including two exons(48bp and 375 bp)and one intron(1371 bp),The transcription start site is located 734 bp upstream of the ATG start codon of MaltObp9 gene.The promoter sequences(-534～-289)were detected by the dual luciferase reporting system,and it was found that the promoter sequences(-534～-289)had strong promoter activity,and there might be cis-acting elements to enhance transcriptional activity.With the promoter sequence(-534～-289)as the target,33 predicted transcription factors were screened using the JASPAR database,which can bind to this region at 16 action sites.Based on comprehensive screening scores,transcription factor functions,expression patterns,and research status,9 transcription factors were finally selected for subsequent verification,including Malt Deaf1,Malt Dll,Malt Ems,Malt Hb,Malt Nub,Malt Pb,Malt Scr,Malt Slou,and Malt Ubx.The transcription factor Malt Ems can participate in the transcriptional regulation of MaltObp9 was verified.By overexpressing the above 9 transcription factor proteins in Drosophila S2 cells,combined with a dual luciferase reporter system,the effects of each transcription factor on the(-534～-289)promoter fragment were detected.The results showed that after the overexpression of transcription factors Malt Deaf1,Malt Ems,and Malt Pb,the luciferase activity detected in the experimental group was extremely significant higher than that of the other control groups(p<0.001),and the transcription factors Malt Deaf1,Malt Ems and Malt Pb had obvious positive regulatory effects on the promoter region of MaltObp9 gene(-534～-289),while the other 6 transcription factors had no obvious regulatory effect on the promoter region.The soluble proteins of Malt Deaf1 and Malt Ems were successfully obtained by prokaryotic expression for EMSA binding experiments,while Malt Pb was expressed in inclusion bodies,and sufficient soluble proteins were not obtained for EMSA verification.The interaction between Malt Deaf1 and Malt Ems and the promoter region was explored by EMSA experiment.The results showed that Malt Deaf1 could not directly bind to the promoter region(-534～-289)of the MaltObp9gene,and Malt Ems could directly interact with the promoter region(-534～-289)through two sites,causing changes in DNA migration rate,showing obvious lag bands,indicating that the transcription factor Malt Ems could participate in the expression regulation of MaltObp9.In conclusion,this study confirmed that MaltObp9 is expressed in the olfactory organ of Monochamus alternatus Hope,has secretory properties,and has the characteristics of universal binding to host plant volatiles,among which Phe54 is the key site for MaltOBP9to bind volatiles.The transcription factor Malt Ems can bind to the upstream sequence of MaltObp9,and has a positive regulatory effect on the expression of MaltObp9.The research results have laid an important foundation for elucidating the molecular mechanism of olfactory perception in Monochamus alternatus Hope.