Molecular Recognition Mechanism Between OBPs Heterodimer In Holotrichia Oblita (Faldermann) And Special Odors

Holotrichia oblita Faldermann(Coleoptera: Melolonthidae)are the key underground insect pests seriously damaging crops such as wheat,peanut,soybean,corn,Chinese sorghum and potato.Managing underground pests with chemical pesticides could lead to many potential threats.Therefore,an environmentally friendly method for controlling of H.oblita is needed.Host plant volatiles are considered to be a good bait to attract and kill the adults.The goal of this thesis is to elucidate the molecular mechanism on cooperative interaction of HoblOBP1 and HoblOBP2 in adult's antennae enhancing the capacity of binding odor compounds.In this study,we first demonstrated the co-expression of HoblOBP1 and HoblOBP2 in the antennae.Then we predicted and analyzed the binding pattern between two proteins and the key binding sites of two protein complexes with four kinds of odors using protein docking software.Specific mutated genes were obtained by site-directed mutagenesis,and these mutant genes were expressed and purified to get different mutated proteins.Then the roles of the different amino acid sites were proved to be involved in the recognition process of HoblOBP1/OBP2 with odors by fluorescence binding assay.The above studies could make clear the mechanism of OBP heterodimer formation and the recognition mechanism of them with odors.The main results are as follows:1.Confirmation of the likely role of heterodimers was demonstrated by fluorescence in situ hybridization.The results showed that HoblOBP1 and HoblOBP2 are co-expressed in some antennal sensilla of H.oblita.2.Using protein docking,we obtained the 3D(dimensional)structure models of Hobl OBP1 and HoblOBP2 complexes.Then we analyzed the the binding pocket of HoblOBP1,HoblOBP2 and HoblOBP1/OBP2 complexes that were formed by 68,77,79 amino acid residues,respectively.Interestingly,the binding pocket of the protein complexes was made up of Hobl OBP1 and HoblOBP2,however,HoblOBP2 was mainly element.3.We further analyzed the binding pocket of HoblOBP1/OBP2 complexes,and discovered 3 pairs of hydrogen bonds,that were formed by the HoblOBP1-Asp61/ HoblOBP2-Tyr86,Hobl OBP1-Gly85/ HoblOBP2-Ile75 and HoblOBP1-Ile87/ HoblOBP2-Asn74,respectively.In the study,the broken of hydrogen bonds within the HoblOBP1/OBP2 complexes were achieved in the form of single point mutation(Gly85 in HoblOBP1,Asn74 and Tyr86 in Hobl OBP2)and double point mutation(Asn74 and Tyr86 in HoblOBP2).The fluorescence binding assay showed after breaking a pair of hydrogen bond or two pairs of hydrogen bonds within the HoblOBP1/OBP2 complexes,the binding curves and scatchard plot of all the protein complexes with 1-NPN,respectively,presented downtrend and kept “J” concave curve downward.The mutant lost almost all of its efficient binding capacity to the four compounds(trans-2-Hexenol,1-Heptanal,R-(-)-Linalool and ?-Terpineol).Only the protein complexes with the mutated site G85 in HoblOBP1 still had weaker affinity with ligands.While three pairs of hydrogen bonds all were interrupted,non-affinity exsited among protein complex and ligands.In addition,the protein complexes with 1-NPN showed uptrend and whose scatchard plot presented the linear correlation.So the 3 pairs of hydrogen bonds played pivotal roles in the process of OBPs binding odors.However,compared to HoblOBP1-Asp61/HoblOBP2-Tyr86 and HoblOBP1-Ile87/HoblOBP2-Asn74,HoblOBP1-Gly85/HoblOBP2-Ile75 may not be the key hydrogen bond.4.The study showed Asn54,Thr71,Asn74,Leu126 and Asn127 in HoblOBP2 were involved in the formation of hydrogen bonds between HoblOBP1/OBP2 complexes and four kinds of ligands.Besides,three hydrophobic residues(Val86 in HoblOBP1,Leu59 and Met117 in HoblOBP2)around ligands were also involved in the process of protein binding ligands.Then,Asn54,Thr71,Asn74,Leu126,and Asn127 in the HoblOBP2 involved in hydrogen bonds were mutated into alanine,respectively.Meanwhile,the two hydrogen bonding sites(Leu126 and Asn127 in HoblOBP2)between complexes and trans-2-hexenol were both mutated into alanine,and the same to the binding sites(the Thr71 and Asn74 in HoblOBP2)of omplex and ?-terpineol.The fluorescence binding assay showed that the five hydrogen bonding sites were mutated into Ala,the two protein complexes had non-affinity with four kinds of ligands,respectively.The protein complexes with the double point mutation lost all of its affinity with ligands.After the hydrophobic residue(Val86 in HoblOBP1)was mutated,the protein complexes showed weaker affinity with ligands.Therefore,the influence of hydrophobic residues were weaker than hydrogen bonds.Our studies elucidated the molecular mechanism on cooperative interaction of HoblOBP1 and HoblOBP2.It is a crucial mechanism on enhanced binding capabilities of heterodimer HoblOBP1/OBP2 and odors in H.oblita because of the 3 hydrogen bonds for heterodimer formation and 5 hydrogen bonds for recognition to odors.Therefore,the results will clarify the molecular recognition mechanism between H.oblita and host plants based on olfactory system,and it is also very important to develop new strategies for adult control of this underground pest.