Molecular Characterizations And Prokaryotic Expression Of Olfactory Related Genes In Grapholita Molesta(Busck)

Throughout evolution, the olfactory system of insect have become highly sensitive,enabling recognize chemical signal from the environment and triggers insect behavioralresponse, such as locating mates, food source, habitat, and ovipositon. Olfactory-basedcontrol strategy has become more and more important in integrated pest management （IPM）.The oriental fruit moth, Grapholita molesta is a severe pest of various fruit crops throughoutworld. Control of G. molesta predominantly relies on chemical pesticides and fruit bagging.However, due to the larvae burrow into fruit and damage only the inner tissues, the chemicalpesticides, which control G. molesta, are not satisfactory. With the increasing prices of laborpower, the fruit bagging is also difficult for orchardists to put it into effect. A strategy forcontrolling adult G. molesta, based on chemical stimuli derived from host plant, is desirableTherefore, it is very important to explore the chemical communication system of G. molesta.With the insights on the chemical communication system, it will be facilitated for design andimplementation of novel monitoring and control strategies against G. molesta.In this paper, the physiological basis of G. molesta chemical communication system,including adult behavioral rhythm, the morphology, structure and distribution of antennalsensillae, was firstly studied. And then, the chemical communication system of G.molesta wasfurther studied at molecular level. In later part, we studied two functional proteins in insectchemical communication system, general odorant binding proteins （GOBPs） andchemosensory proteins （CSPs）. The main results are as follows:1. The behavioral rhythms of adult Grapholita molestaIn the laboratory, the Behavioral characteristics of G. molesta，including adult emergence,mating and oviposition behaviors, were investigated under a L15:9D regime at24±0.5℃and70±10%r.h. The results revealed that90%of adults emergence occurred between05:00and10:00, the peak of emergence was between05:00and06:00；the calling behavior of adultscommenced in the3rd days after adult emergence. males and females mated with copulatorypose of “一” type. The mean duration of mating was22.07min. and more than90%ofmating occurred between17:00and21:00；Oviposition in most of females occurred between 17:00and21:00amounting to87.43%of total oviposition.2. Ultrastructural observation of antennal sensilla of adult GrapholitamolestaThe antennae of G. molesta were observed by scanning electron microscopy. The resultsindicated that the antennae of G. molesta were threadlike and made up of scape, pedicel andflagellum. There were seven kinds of sensillae, including sensilla trichodea, sensillabasiconica, sensilla chaetica, sensilla styloconica, sensilla coeloconica, sensilla auricillica andB hm bristles.3. Cloning and characterization of GOBP genes from Grapholita molestaTwo new general odorant binding protein （GOBP） genes, GmolGOBP1andGmolGOBP2, were cloned from antennae of female G. molesta by use of RT-PCR and RACE.The GenBank accession numbers are JN857939and JN857940, respectively. The full-lengthcDNA of GmolGOBP1is1090bp with a492bp open reading frame encoding164aminoacids. GmolGOBP2cDNA is637bp in length containing a483bp open reading frameencoding161amino acids. Proteins signature analysis indicated that they contains anN-terminal signal sequence of20amino acids. The two GOBPs from G. molesta show lowsequence identity （48%）, but they are similarity to GOBPs of the same group from otherlepidopteran, suggesting that the two GOBPs belong to different classes of lepidopteranGOBPs.RT-PCR analysis indicated that GmolGOBP1and GmolGOBP2are only expressed inantennae of both sexes. Real-time PCR analysis further revealed that the transcript level ofGmolGOBP1was higher in males than in females, whereas the transcript level ofGmolGOBP2was higher in females than in males. The high transcript levels of GmolGOBP1in both sex and GmolGOBP2in female were detected at the end of photophase and duringscotophase. The expression of GmolGOBP2in male remained at similar levels during thecomplete photoperiod. Based on these results, the possible physiological functions ofGmolGOBPs are discussed.4. Cloning and sequence analysis of CSP gene from Grapholita molestaA novel chemosensory protein gene, GmolCSP, was cloned from antennae of adult G.molesta by use of RT-PCR and RACE. The GenBank accession numbers are JQ821389. Theisolated cDNA encoding GmolCSP contains a384bp open reading frame for a polypeptide of127amino acids. The predicted amino acid sequences contain an N-terminal signal sequence of18amino acids. The mature deduced GmolCSP consist of109amino acids with amolecular mass of12.80kD, and an IP of8.33. after analysis of sequence similarity andphylogenetic tree, it was indicated that GmolCSP share high homology with CfumCSP3（73%）, and then HvirCSP2（68%）, however, GmolCSP share low similarity with otherlepidopteran （34～46%identity）. RT-PCR analysis indicated that GmolCSP expressed in allof the test tissues, including antenna, head, head without antennae, thorax, abdomen, leg andwing.5. The prokaryotic expression of GOBP2and CSP from Grapholita molestaHeterologous expression of genes can provide insights to the gene function. Based oncloned GmolGOBP2and GmolCSP information, the GmolGOBP2and GmolCSP wereconstructed into prodaryotic expression vectors pET-32a, and expressed in BL21（DE3）.The results of Western Blot indicated that the recombinant prteins （pET/GmolGOBP2andpET/GmolCSP）were successfully expressed. Our results are not only helpful for the researchof GmolGOBP2and GmolCSP, but also can form a solid basis to explore the chemicalcommunication system of G. molesta.