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Functional Analysis Of Related Olfactory Genes In Oriental Fruit Moth,grapholita Molesta Busck

Posted on:2019-03-24Degree:DoctorType:Dissertation
Country:ChinaCandidate:X L ChenFull Text:PDF
GTID:1363330596955122Subject:Agricultural Entomology and Pest Control
Abstract/Summary:PDF Full Text Request
The oriental fruit moth,Grapholita molesta?Busck?,is a notorious pest of stone and pome fruits throughout the world,causing serious losses in fruit yields.In the area of mixed cultivation of peach and pear,oriental fruit moth has obvious behavior of a host shift.In the spring to midsummer?the first three generations of G.molesta?,they mainly injury peach shoot,then move to orchard and destroy pear(the 4th to 5th generations).Identification of molecular mechanism from host plant volatiles by G.molesta is important to the development of ecological regulation technology based on the plant source attractant.Antennae are the primary olfactory organs of insects,and there are many sensilla on the antennae.A diverse range of olfactory proteins,including odorant-binding proteins?OBPs?,chemosensory proteins?CSPs?,olfactory receptors?ORs?,ionotropic receptors?IRs?,sensory neuron membrane proteins?SNMPs?and odorant degrading enzymes?ODEs?are immersed in the sensillum lymph.OBPs are small,water-soluble globular proteins that could selectively bind and transport the hydrophobic odorants in the external environment through the hydrophilic lymph to the odor receptors located on the sensory neuron membranes,and then triggered signaling and subsequent behavioral responses.OBPs play the role as"identity inspector"and"transporter"in the insect olfactory system to ensure only small number of odorous molecules that are active to specific insects in the environment could enter the interior of the sensory apparatus.ORs were more specific in the identification of odor molecules,which could transfer chemical signals from the active odor molecules into electrophysiological signals and trigger signal transduction and subsequent processing.ORs and OBPs play crucial roles in olfactory communication of insects.The physiological functions of OBPs and CSPs with high abundance expression in the antennae of G.molesta have been studied.In this research,the binding function of OBPs with low abundance or non-antenna specificity was analyzed,to clarify the roles of these proteins in identifying specific components or trace components of host plant volatiles.Moreover,RNAi technology was used to study the physiological function of co-receptor in the identification of volatiles.The main results are as follows:The complete coding sequences of eight odorant binding proteins were cloned and sequenced based on transcriptome of the female G.molesta antenna?Genbank No.GmolPBP1,MF066363;GmolOBP4,MF066357;GmolOBP5,MF066358;GmolOBP7,MF066359;GmolOBP10,MF066360;GmolOBP12,MG581971;GmolOBP14,MF066361;GmolOBP16,MF066362?.The multiple sequence alignment and phylogenetic tree analysis showed that there were only 4 conserved cysteine residues in GmolOBP14,and clustered to the subfamily of Minus-C OBP independent evolutionary branches,indicating that GmolOBP14 belonged to Minus-C OBP.Other OBPs had six conserved cysteine residues,belonging to typical Classic OBP subfamily genes.Compared with other OBPs,GmolOBP7belonged to long OBPs,the coding region was 504 bp and encoding 167 amino acids.Signal peptide analysis revealed that there was no signal peptide at the N-terminal of GmolOBP7.To further identify whether there was a signal peptide at N-terminal of GmolOBP7,the DNA fragment of 281 bp was obtained using 5'RACE amplification at the 5'end of coding region,no signal peptide at the N-terminal of this protein was confirmed using Signal P.The qRT-PCR results showed that GmolPBP1,GmolOBP4,GmolOBP5,GmolOBP10,GmolOBP12 and GmolOBP16 were mainly expressed in antennae of male and female moth.GmolOBP7 was highly expressed in male wings and antennae of both sexes,but the expression levels of GmolOBP7 in male antennae and female antennae were not significant.GmolOBP14 was most highly expressed in male wings,about 6.98 folds and 2.84 folds to male and female antennae,respectively.The expression levels of GmolOBP14 in male wings were significantly higher than the antennae of male and female moth.It is speculated that GmolOBP14 was not only involved in olfactory physiological function but also has the taste function of recognizing non-volatile host metabolites.Expression profiles of GmolOBPs were different between male and female.For example,the expression levels of GmolPBP1,GmolOBP4,GmolOBP5 and GmolOBP12 in male were 9.74,1.73,2.46 and 2.92 folds than the female antennae,respectively.The results indicated that the functions of the six GmolOBPs in male and female antennae were different.Eight recombinant GmolOBPs were successfully expression in E.coli prokaryotic expression system.GmolOBP7 existed in soluble form.The other recombinant GmolOBPs existed in the form of inclusion bodies.Active fusion proteins were obtained by denatured,refolding and protein purification.The results of fluorescence competitive binding assays suggested that GmolOBP4,GmolOBP12 and GmolOBP16 could strongly bind the pheromone cis-8-dodecene 1-alcohol,with the Ki values of 6.19,2.73 and 7.76?M,respectively.GmolOBP7 and GmolOBP10 showed binding activity to the secondary pheromone components,1-dodecanol?12:OH?,with the Ki values of 9.27 and 5.10?M,respectively.GmolOBP5 and GmolOBP14 showed no binding activity to the 4 sex pheromones,indicating that they were not involved in the perception and recognition of sex pheromones.The competitive combination of seven GmolOBPs to more than 30 peach and pear volatiles showed that the binding capacities of GmolOBP4 to the candidate odorant ligands were weak,with moderate binding activity of hexaldehyde?Ki=16.60 M?and pear ester?Ki=15.75?M?,binding spectrum of the GmolOBP5 was narrow and GmolOBP5 could only weakly bound 9 ligands of the putative 35 species.GmolOBP7 showed strong binding activity to dodecals,pear esters and?-Ocimene and with the Ki values of 6.21,2.75 and 9.52?M,respectively.The RNAi experiment was conducted to further confirm that GmolOBP7played an important role in male recognition of secondary pheromone 12:OH and pear ester?a volatile component of host plants?.GmolOBP10 showed strong binding capacities to1-hexanol,1-decyl alcohol and?-Ocimene,with the Ki values of 5.19,9.92 and 10.89?M,respectively.The binding capacity of GmolOBP12 to trans-2-hexenal,benzaldehyde,1-hexanol and acetic acid-cis-3-hexenyl ester was the strong,with the Ki values of 4.21,6.81,4.68 and 4.68?M in turn.GmolOBP14 and GmolOBP16 displayed relatively weak in binding affinities to other volatiles except pear esters.1-hexanol is an important green leaf volatile releasing from peach and pear fruit;it was found that GmolCSP8 could specifically bind to 1-hexanol.In this study,the molecular mechanism of GmolCSP8 binding to 1-hexanol was further studied by using homology modeling,molecular flexible docking and fixed-point mutation.At first,the 3D structure model of GmolCSP8 was constructed based on the crystal structure of desert locust SgreCSP4,it was found that Thr27,Leu30,Val40,Leu80 and other residues could be important sites for binding to 1-hexanol using molecular flexible docking.After Thr27 was mutated into Ala27,the mutant GmolCSP8-T27A loosed the ability to bind to 1-hexanol,suggesting that Thr27was a key site for GmolCSP8 binding to 1-hexanol.When the Leu30 was mutated into Ala30,the binding capacity of mutant GmolCSP8-T27A to 1-hexanol was reduced by about 6 folds,implying that Leu30 had significant effect on GmolCSP8 binding to 1-hexanol.After Val40and Leu80 were mutated into Ala40 and Ala80,the binding capacities of mutant proteins to1-hexanol did not change significantly,suggesting that Val40 and Leu80 were not the key amino acid residues in GmolCSP8 binding to 1-hexanol.The complete coding sequence of GmolOrco was obtained by RT-PCR using antenna cDNA of female G.molesta?Genbank No:GmolOR2,MG581972?.Multiple sequence alignment showed high homology between GmolOrco and other insect Orco.GmolOrco had the higher degree of identical with CpomOrco and LbotOrco at 96%and 91%,respectively.Evolutionary analysis showed that Orco of different insect species were all clustered into independent evolutionary branches,suggesting that they were evolutionarily homologous.The expression patterns of GmolOrco in different tissues of adult and development stages displayed that GmolOrco was predominantly in antennae of both sexes,and the expression level of male antenna was significantly higher than that of female?P<0.05?.The GmolOrco was expressed during all developmental stage,that is,from egg to adult stages.GmolOrco was most highly expressed in male antennae,and was significantly higher than female antennae.The expression level of GmolOrco in egg and 5-day-old pupa was also higher.The olfactory function of GmolOrco was studied using RNAi and electroantennogram?EAG?,the results displayed that the EAG response values of dsGmolOrco-treated male to sex pheromone Z8-12:Ac and A8-12:OH were significantly decreased compared with the dsGFP-treated and the non-injected group?P<0.05?,suggesting that GmolOrco was involved in male recognition of female sex pheromones.EAG response values of dsGmolOrco-treated female to the host plant volatiles acid-cis-3-hexene ester decreased to 47.74%,indicating that GmolOrco also had the function of identifying the host plant volatiles in the female.
Keywords/Search Tags:Grapholita molesta, odorant binding protein, chemosensory protein, odorant receptors, fluorescence competitive binding test, RNA interference
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