Identification And Functional Analysis Ofthe Olfactory Genes In Holotrichia Parallela(Coleoptera: Melolonthidae)

Underground pests are a harmful class group in agricultural industry in China. The scarab beetle, Holotrichia parallela Motschulsky(Coleoptera: Melolonthidae), belongs to such a class and has caused serious economic damage to crops, fruit trees and forest trees in China. The sophisticated olfactory system plays a crucial role in insect survival and adapt ation to the environment. And it helps insects to detect and discriminate between different kinds of odorants, which are volatile small organic molecules in the environment. These cues guide the insect towards escaping behaviors, seeking a mate, mating, ovipositing, host location of food and habitat. H. parallela perceive and distinguish amounts and kinds of odorants between individual, and between different species of insects and their host plants odors, and produce related behavioral reactions. The study on H. parallela olfactory receptive mechanism and related olfactory prorein, help to reveal the secret of the process. The futher understanding may provide reliable target sites for the development of efficient insect trapping agent and repellent, to control pest protection, and achieve the purpose of pest control protection, gain a new avenues for the biological control. This study identified related olfactory prorein gene family, and the odorant binding protein Hpar OBP15 a were cloned and tested the binding affinities. Our conclusions are listed as follow.1. Deep sequencing and assembly of H. parallela antennal transcriptome yielded 58,763 transcripts with an average length of 879 bp and identified some related olfactory prorein, including 24 odorant binding proteins(OBPs), 8 chemosensory proteins(CSPs), 4 sensory neuron membrane proteins(SNMPs), 28 odorant receptors(ORs) and 10 ionotropic receptors(IRs). Among them, the genes that 20 Hpar OBPs, 7 Hpar CSPs, 1 Hpar SNMP, 21 Hpar ORs, 6 Hpar IRs are with the complete open reading frame(ORF). We identified 3 Plus-C OBPs(Hpar OBP14, 16, 39) and 4 Minus-C OBPs(Hpar OBP1, 12, 27, 29) and the rest of Hpar OBPs contain the complete 6 conservation cysteine. All 8 Hpar CSPs contain a signal peptide, 4 conserved cysteine and similar two-stage structure. We also found that 26 Hpar ORs roughly divided into group1,group2 and group3 subfamily. 7 Hpar IRs is considered antennal specific genes in H. parallela.2. Based on transcriptome sequencing of the adult antenna of H. parallela, reverse transcription-polymerase chain reaction(RT-PCR) was used to amplify the ORF of Hpar OBP15 a. The m RNA levels of Hpar OBP15 a in different tissues of adults were assayed by real-time quantitative PCR(q RT-PCR). The full length c DNA of Hpar OBP15 a is 534 bp( Gen Bank accession No.: AK1834747), encoding 147 amino acid with a predicted molecular m ass of 15.7 k Da. Hpar OBP15 a m RNA was specifically expressed in the antennae of female adults, only trace expression in the head, chest, legs and wings.3. Binding affinities of Hpar OBP15 a towards 58 compounds, which included compounds from volatile green plants and putative sex pheromones of H. parallela, were measured by fluorescent competitive binding assay. Among the 58 chemicals tested, Hpar OBP15 a exhibited a good affinity with 46 kinds of ligands. Notably, it was found that Hpar OBP15 a had the strongest binding capacity to dodecane and 1-dodecanol, with the dissociation constant of 8.5 μmol/L and 11.3 μmol/L, respectively. Hpar OBP15 a had a certain binding specificity toward the sex pheromone components methyl L-isoleucinate and(-)-linalool, with the dissociation constant of 21.0 μmol/L and 18.5 μmol/L, respectively. Hpar OBP15 a protein has a strong specificity toward dodecane, a volatile from host elm leaves. This protein may play an important role in locating host of H. parallela.