Functional Analysis Of Odorant-binding Proteins In Anomala Corpulenta Motschulsky(Coleoptera: Scarnaeoidea)

Anomala corpulenta Motschulsky is a predominant underground pest in the whole China As one ofthree dominant Scarab Beetle species (scarabeidae family) and caused substantial amount of losses todifferent kinds of crops and fruit trees. Larvae are live in soil and seriously damage underground parts ofplants or near-surface parts of the plants, such as roots or stem of seedlings. That damage result indeclining of both quantity and quality of final yield.Insect antenna as the organ to senses the environment is the essential part of olfactory system is playsa crucial role in the process of food finding, mating, and choosing a site for oviposition. There are manykinds of olfactory sensilla in antenna and sensilla placodea and sensilla are in the majority. Odorantbinding proteins (OBPs) are the first functional proteins which involved in the olfactory perception. OBPsare believed to be responsible for the binding and transport of odours from the environment to ORs locatedon the membranes of olfactory neurons in the early stages of insect odorant receptionIn order to further understand the olfactory mechanism of A. corpulenta and control them by scientificmethods, we identified two the odorant-binding proteins of A. corpulenta by cDNA library and researchtheir function. The primary results are as follows:(1) In order to study the function of the olfactory related proteins in A.corpulenta, a full-length cDNAlibrary of A. corpulenta antennae was constructed based on SMART technique. Primary library titer was6.74×106pfu/mL and amplified library showed a titer of1.08×1010pfu/mL. The recombinant rate was98.97%and the sizes of amplified fragments were0.5~2.0kb, with an average of1.0kb.（2）Two full-length odorant-binding protein sequences named AcorPBP1, AcorPBP2were identifiedand Genbank numbers are KF445134and KJ093447. Using softwares Clustal and MEGA4.0to analyzethe sequences, we can explore the genetic relationship between, Anomala corpulenta OBPs and otherColeoptera insects order.（3）AcorPBP1and AcorPBP2were cloned and expressed in Escherichia coli. After purifying thetarget protein, we tested the binding ability of the two PBPs to31compounds with fluorescence probe1-NPN. The result show PBP1and PBP2have broader binding properties to all of the volatiles exceptβ-caryophyllene. Ten different volatiles, β-lonone, hexyl benzoate, α-lonone, nerolidol,1-heptanol,2-tridecanone,1-undecanol,1-nonaol,1-hexadecanolcety,2-cyclohexen-1-one, showed good affinity to the two odorant-binding proteins in binding assays and the Ki is less than10μM. The affinities ofAcorPBP1to aliphatic alcohols are higher than aliphatic terpenoids, but AcorPBP2is contrary.Interestingly, we observed that their binding capacity was increased when the number of carbon atoms inthe carbon chain added before exceeding eleven.