| Odorant binding proteins(OBPs)could bind and transport odorants specifically,which is the first part of olfactory sensory system.And using OBPs as molecular targets for the screening of insect behaviorally active compounds is one of the main research contents in reverse chemical ecology.Present study shows that the fluorescence competitive binding assays present the binding abilities but lack more characteristic analysis including important conformational transformation,which hinders the research of binding mechanism of OBPs.Meanwhile,Insects orderly regulate the expression of different OBPs to perform different olfactory recognition functions in different developmental status,which is the important basis of specificity of the olfactory system.Transcriptional regulation is the first step in gene expression regulation.However it has not been reported that any transcription factors could act on the transcription of OBPs gene,which leads the lack of molecular interpretation in molecular level for the synergistic effect of OBPs and the relationships between OBPs expression with insect development and environmental change.These problems limit the further development of reverse chemical ecology targeting OBPs.Therefore,in this research,by Monochamus alternatus Hope and Dastarcus helophoroides(Fairmaire)as the research object,DhelOBP21 from Dastarcus helophoroides,which high expressed in antennae,was investigated in the aspect of the ligand-binding mechanism and MaltOBP19,which has been defined the olfactory function,was studied the transcriptional regulation mechanism.The results of this research will provide theoretical foundation for binding mechanism and transcriptional regulation mechanism of OBPs and biomolecular evidence for the screening of insect behaviorally active compounds,which also provide new target for pest management based on olfactory control.The main findings are as follows: 1 Ligand-binding mechanism for DhelOBP21 from Dastarcus helophoroidesThe full–length cDNA encoding DhelOBP21 was cloned.The unmated adults expressed significantly highest levels of DhelOBP21 in their antennae and such expression decreased at mating and oviposition stages.The results of molecular modeling and molecular docking showed that DhelOBP21 had a hydrophobic binding cavity at the center of the protein core.Most area of cavity walls was hydrophobic,while one area was polar formed by Ser67(number 67 after cut the signal peptide).To alter the hydrophobicity of the binding cavity,Ser67,Ile84 and Thr119 were mutated to Ala,Asn and Asn,respectively.The first mutation aimed to increase hydrophobicity,while the other two mutations were designed to decrease the hydrophobicity.Ligand-binding experiments showed that molecular volume should be considered at the beginning of binding.Hydrophobic interactions and van der Waals force were the major interaction force and hydrogen-bonding interactions was of less effect on the enhancement of ligands.The binding of protein and ligands depended on the whole of cavity amino acids instead of single amino acid.And the volume and shape changed because of the mutation of a single amino acid,which lead the orientation of ligands and interactions changed,and finally influenced the binding affinities.By contrast,at pH 5.0,DhelOBP21 showed a weaker binding ability against most ligands,while the mutant protein S67 A was in opposite ways.The Ser67 residue may play an important role in the release of ligands.2 Using DhelOBP21 as molecular targets for the screening of insect behaviorally active compoundsBased on the binding mechanism of DhelOBP21 with ligands,fluorescencequenching assays and circular dichroism(CD)were used to measure more binding character and structural transformation of DhelOBP21.The quenching mechanism of DhelOBP21 bound to Butylated hydroxytoluene,Camphor,(+)-?-pinene,(-)-Fenchone and Myrcene were static,which indicates that stable complex formed.While the quenching mechanism of DhelOBP21 bound to others were dynamic,which indicates that the quenching were lead by molecular collision and no complex formed.CD spectra showed DhelOBP21 solution exhibited the characteristic of a protein with high ?-helical content.At pH 7.4,some ligands could lead to the increase of ?-helical content,which were coincident with atom molecular dynamics of DhelOBP21.In the binding process with some ligands,the amino-terminus transformed conformation form random coil to ?-helix and the new ?-helix covered the binding cavity like a lid.Comprehensive judgments are made on the basis of two factors: whether different ligands can form stable complexes with proteins and whether they can induce the increase of alpha helix content.(+)-?-pinene was selected to test the behaviorally active by Y-tube olfactometer bioassay.D.helophoroides unmated adults showed a significant preference for(+)-?-pinene.After RNAi experiments,the adults lost the behavior reaction,which indicates that DhelOBP21 is the pivotal recognition protein for(+)-?-pinene.3 Transcriptional regulation mechanism in MaltOBP19 gene form Monochamus alternatusThe promoter region upstream(948 length)of MaltOBP19 gene were cloned from the Monochamus alternatus Hope.The cis-acting elements in the known MaltOBP19 promoter sequence were predicted with the JASPAR database.There were total 38 predicted cisacting elements with 100% identity with the database in the MaltOBP19 promoter region,which were belonged to 10 transcription factors.The activity of 5'progressive deleted MaltOBP19 promoter were figured out by Dual-luciferase assays in Drosophila S2 cells,which showed that the sequence region of-515 to-312 contained the positive regulatory cis-acting elements.Further,the region of-474 to-433 contained the negative regulatory cis-acting elements,while the region of-349 to-312 contained the positive regulatory cisacting elements.These results predicted complicated regulatory mechanism.Based on predicted results of JASPAR database and the researches of transcription factors,the transcription factor BarH1 was selected to detect transcriptional regulatory activity in MaltOBP19.The full–length cDNA encoding MaltBarH1 was cloned.Taken advantage of Dual-luciferase assays and eukaryotic expression vector,MaltBarH1 was over-expressed in S2 cells.Comparing with control group,the promoter activity of the region of-405 in the promoter of MaltOBP19 gene was down-regulated by the overexpression of MaltBarH1.Recombinant soluble proteins of MaltBarH1 were obtained in vitro,which were prepared for EMSA.The results showed that recombinant soluble proteins could bind and shift the probe.And the upper complex was competitively inhibited by the presence of the unlabeled probe,which indicated the specific protein: DNA interaction.Based on the above results,transcriptional regulatory activity of MaltBarH1 in MaltOBP19 promoter was proved preliminary.In summary,more binding characteristics and conformational transitions analysis of OBPs are necessary for olfactory function and screening of insect behaviorally active compounds,which contributes to accurate data and the improvement of screening efficiency.MaltBarH1 has been confirmed the transcriptional regulatory activity in MaltOBP19 gene preliminary,which lays the biological foundation for the relationship between expression of OBPs gene and olfactory function.And with the development of RNAi technology in pest management,targeting transcription factors of OBPs gene will be a new strategy for olfactory control. |
PDF Full Text Request