Binding Characteristic Analysis Of OBPs In Aenasius Bambawalei

P.solenopsis is a seriously invasive mealybug and it has resulted in serious damage to cotton in the world.A.bambawalei Hayat is a dominant endoparasitoid on P.solenopsis,and it would be contributed to biology control of the mealybug.Odorant binding protein of insect plays key role in binding and transporting hydropbic odorants from the environment to odorant receptors.It's essential to reveal the binding and reseasing mechanism between AbamOBPs and ligands for screening behavior active compounds of A.bambawalei.In this study five genes of A.bambawalei were choose and cloned successfully.The binding characteristics between AbamOBP50 and 22 host volatileswas measured by using a fluorescence competition-binding assay.Besides,the interaction of AbamOBP1 and ligands were measured by using fluorescence quenching,Circular Dichroism and Molecular modeling and docking.In addition,the function of AbamOBP1 in host location by A.bambawalei was tested by RNA inteference and Y-tube olfactometer.The main results are as follows: 1.Cloning,expression and purification of AbamOBPs from A.bambawaleiAbamOBP50,OBP47,OBP43,OBP41 and OBP1 genes of A.bambawalei were successfully cloned by RT-PCR.The open reading frame of AbamOBP50,OBP47,OBP43,OBP41 were 369 bp,378bp,405 bp,402bp and 408 bp,They encoded 123,126,135,134 and 136 amino acids respectively,The length of signal peptides were 21,21,17,23 and 20 amino acid residues respectively.AbamOBP50,OBP1 and OBP41 belonged to Classic OBPs,OBP47 belonged to Plus-C OBPs,OBP43 belonged to Minus-C OBPs.We successfully constructed the recombinant plasmids pET30a-AbamOBP50 and pET17b-AbamOBP1.pET30a-AbamOBP50 and pET17b-AbamOBP1 were mainly expressed in inclusion bodies.AbamOBP50 and AbamOBP1 proteins were purified by Ni sepharose high performance column and DE52 affinity columns respectively.2.The binding affinity of AbamOBP50 from A.bambawalei1-NPN was choosed as a fluorescent probe.The binding properties between AbamOBP50 and 22 kinds of plant volatiles were measured at neutral and acidic pH by fluorescence competitive binding assays.AbamOBP50 showed highest binding affinities within all volatiles at neutral pH,and it showed high binding affinities with eight compounds at acidic pH,including 1-Octen-3-one,2,4,4-TriMethyl-2-pentene,S-(-)-limonene,Myrcene,?-Phellandrene,?-Caryophyllene,Camphene and Nerolidol.However,it showed higher binding affinities at acidic pH than at neutral pH with Myrcene,S-(-)-limonene,?-Phellandrene and ?-Caryophyllene.3.Fluorescence quenching assay of AbamOBP1 from A.bambawaleiWe tested the Fluorescence quenching between AbamOBP1 and four plant volatiles 1-Octen-3-one,1-Octen-3-ol,2,4,4-TriMethyl-2-pentene and S-(-)-limonene.The results showed that the quenching of AbamOBP1 by 1-NPN is static at pH7.4 and pH5.0,AbamOBP1 combined with 1-NPN by electrostatic interactions at pH5.0,by Hydrophobic at pH7.4,the stoichiometry of the binding of AbamOBP1 to 1-NPN was 1:1.The static quenching occcurred between AbamOBP1 and 1-Octen-3-one,2,4,4-TriMethyl-2-penten,1-Octen-3-ol at pH7.4 and pH5.0.Static quenching occurred between AbamOBP1 and S-(-)-limonene at pH7.4,but dynamic quenching at pH5.0.AbamOBP1 combined with 1-Octen-3-one,2,4,4-TriMethyl-2-penten and 1-Octen-3-ol by Hydrophobic at pH7.4 and pH5.0,The main acting force is Hydrogen bonding between AbamOBP1 and S-(-)-limonene at pH7.4.AbamOBP1 binding 2,4,4-TriMethyl-2-penten 1-Octen-3-ol and S-(-)-limonene may be based on dimer.AbamOBP1 binding l-Octen-3-one may be based on dimer,trimer and polymer.4.Circular Dichroism assay of AbamOBP1 from A.bambawaleiThe CD spectra of AbamOBP1 and that of AbamOBP1 with ligands were measured to investigate conformational changes by using a JASCO-1500 CD Spectropolarimeter.The results showed that two negative dichroic minima at 208 nm and 222 nm at pH7.4,which is characterictic of AbamOBP1 with higher ?-helical content at pH7.4,next at pH3.0,with lower ?-helical content at pH5.0.The molar concentration ratios of AbamOBP1 and four ligands were 1:1,1:3,1:5 and 1:10 at pH7.4.The molar concentration ratios of AbamOBP1 and four ligands were 1:1 and 1:3 at pH5.0 and pH3.0.When four ligands were added to solutions of AbamOBP1,the continuous increased in the negative ellipticity of the 208 nm and 222 nm,which instruct that the ?-helical content were decreased in the three different pH conditions.The binding of AbamOBP1 and ligands lead to the protein peptide chain extension,caused the changed of protein secondary structure.5.The function of AbamOBP1 in host location of A.bambawaleiWe test the behavior choice of males of A.bambawalei to the four cotton volatiles 1-Octen-3-one,1-Octen-3-ol,2,4,4-TriMethyl-2-pentene and S-(-)-limonene by Y-tube olfactometer.The results showed that males of A.bambawalei showed significant selectivity to 1-Octen-3-one and 2,4,4-TriMethyl-2-pentene,However,the males of A.bambawalei showed no selectivity to S-(-)-limonene and 1-Octen-ol.The mRNA expression level of AbamOBP1 was significantly decreased at 48 h and 72 h after the injection of double-stranded RNA(dsAbamOBP1).Then we performed a Y-tube olfactometer at both time points,the preference of males of A.bambawalei for 1-Octen-3-one and 2,4,4-TriMethyl-2-pentene was undetected after the injection of dsAbamOBP1.6.Molecular modeling and docking of AbamOBP1 from A.bambawaleiASP1(3CAB pH7.0,3CDN pH4.0)from Apis mellifera was selected as the template of AbamOBP1 for homology modeling.AbamOBP1 has six ?-helices and three disulphide bridges according to the predicted 3D structure,which belongs to Classi C OBPs.A much smaller binding pocket was observed in AbamOBP1-pH7.0.Molecular docking showed that the residues of lle3,Phe51,Phe54,Leu56,Leu63,Val68,Arg71,lle72,Gly80,Met83,lle84,Phe106,Lys113,Tyr114,Phe115,Val116 and lle117 were involved in the combination of AbamOBP1 and ligands at pH4.0.And the residues of Phe51,Leu56,Leu57,His62,Leu63,Asp64,Lys67,Val68,Leu69,Arg71,Ile72,Ala99,Leu102,Val103,Phe106,Lys113,Tyr114,Phe115 and Val116 were involved at pH7.0.