Cloning, Prokaryotic Expression And Functional Analysis Of Pheromone Binding Protein Relative Genes From The Oriental Fruit Moth, Grapholita Molesta (BUSCK)

The morphology, structure and distribution of sensilla on larval antennae and mouthpartand imaginal proboscis and labial palp were studied in the oriental fruit moth, Grapholitamolesta by using scanning electron microscopy. We also cloned two pheromone-bindingprotein genes and a general odorant-binding protein using reverse transcriptase-polymerasechain reaction (RT-PCR) and rapid amplification of cDNA ends, which characteristic werefurther researched by expression and fluorescence binding assays. The primary results are asfollows:1. The antennae of G. molesta possess two sensilla chaetica, six sensilla basiconica, andone sensillun styloconicum. The labrum bears six pairs of sensilla chaetica. The epipharynxcarries three pairs of flatten sensilla chaetica, three pairs of small epipharyngeal sensilla, andone pair of broad sensillum digitiformium. Each dentate mandible carries two sensillachaetica basally on outer face. On the maxilla, both cardo and stipes carry one sensillumchaeticum. Each galea has two elongate flattened sensilla basiconica, three short-sharpsensilla basiconica, two large sensilla styloconica and one sensilla chaetica. The distalsegment of the maxillary palp possesses seven sensilla basiconica, one sensillun styloconicum,laterally one sensillum digitiformium and two sensillum placodeum. Each labial palp bearsdistally one cone-shaped sensillum chaeticum and one elongate sensillum styloconicum. Inaddition, the function of these sensilla was discussed by comparing them with those of otherinsect larvae.2. Five types of sensilla were identified on the proboscis of G. molesta: sensilla trichodea,sensilla chaetica, sensilla basiconica (two subtypes), sensilla styloconica (two subtypes), andsensilla ampullaceous. Three types of sensilla were found on the surface of the labial palp:sensilla chaetica, sensilla squamiformia, and sensilla campaniformia. In addition we foundsmall holes sporadically located on the walls of the second segment of each labial palp. Thepit organ, which is located on the terminal segment of each labial palp, was found to be86.61μm deep with a21.90μm wide opening. Each pit structure contained three types ofsensilla: furcate and rare furcate sensilla trichodea and mastoid sensory pegs, which were located on the upper half of the pit, and leaf-shaped sensory pegs which were located on thelower half of the pit.3. In this study, we cloned two pheromone-binding protein genes, GmolPBP2(GenBank:KF365878) and GmolPBP3(GenBank: KF365879). They contain510bp and495bp openreading frame that encoded a169and164amino-acid protein, respectively. The deducedamino-acid sequences were characteristic of the odorant binding protein family, namely sixconserved cysteine residues. Genomic DNA sequence of each gene contained two introns,however, the lengths and positions of two introns were different. GmolPBP genes were onlyexpressed in antennae of female and male moths, the transcription level of GmolPBP3in malewas only57.0%compared to those of female, but GmolPBP2had similar amounts (100:96.6)in female and male antennae. The transcription levels of GmolPBP3compared withGmolPBP2were9.0%and5.3%in female and male antennae, respectively. The transcriptlevels of both genes were significantly different in premating and post-coitum individuals,implying that mating affected pheromone response.4. To better understand the functions of GmolPBPs, two GmolPBPs were expressed inEscherichia coli. Fluorescence binding assays showed that GmolPBP2had strongly bindingaffinities with two sex pheromone components E8-12: Ac and Z8-12: Ac, followed by Z8-12:OH and12: OH, and also bound some ordinary odor molecules. However, the affinity ofGmolPBP3with both sex pheromones and ordinary odor molecules was very weak. Theseresults showed that GmolPBP2play main role in pheromone discrimination and recognition.Electroantennogram result showed male moths more sensitive to anabgs of sex pheromonethan female moths. Z8-12: Ac elicited the strongest response to male moths among four sexpheromone, followed by E8-12: Ac and Z8-12: OH, however12: OH without luring to malemoths.5. In this study, a novel general odorant-binding protein cDNA from G. molesta (Busck)was cloned and named as GmolOBP3(GenBank: KF395363). GmolOBP3contains a492bpopen reading frame encoding163amino-acid residues, the predicted molecular weight andisoelectric point was18.62kDa and4.93, respectively. The mature protein of GmolOBP3includes six conservative cysteine residues, which are the hallmark of insect OBPs.GmolOBP3was expressed in the antennae and abdomen of female and male. In five daysafter eclosion, the expression level of female moth GmolOBP3was increased with emergencedays, but5thday the expression quantity of male significantly reduced. The fluorescencecompetitive binding assay indicated that pheromone components in addition of E8-12：Ac and12：OH also had different affinity with GmolOBP3, and the dissociation constant of Z8-12: Acand Z8-12：OH was83.00μmol/L and103.70μmol/L, respectively. GmolOBP3could weak bind with16plant volatiles in which GmolOBP3has the strongest affinity to β-lonone withthe dissociation constant49.36μmol/L. We so inferred that GmolOBP3protein havecharacteristics of selective binding with various ligands.