Identification And Function Of Olfactory Proteins From Daktulosphaira Vitifoliae (Hemiptera: Phylloxeridae)

Daktulosphaira vitifoliae(Fitch)(Hemiptera:Phylloxeridae)is an important quarantine pest of grapes in China.The larvae and adults suck juices from host plants,and damage leaves and roots inducing both radicicoles and gallicoles.In China,grape phylloxera primarily damages the roots of the plants,causing the root rot,and sometimes causes the death of the entire plant.Because this pest primarily lives underground,the olfactory system plays an important role in the selection of host plants.Olfactory proteins are part of their peripheral olfactory system and serve as the molecular basis of this system.The identification and functional analysis of olfactory proteins is necessary to elucidate the molecular mechanisms of this insect's olfactory recognition system and could provide a theoretical basis for biological control of the insect using olfactory disruption of host finding.Therefore,this study on the grape phylloxera olfactory proteins provides a foundation for the future development of olfactory control techniques.We examined olfactory sensilla and identified 27 olfactory proteins by transcriptome analysis in grape phylloxera,conducted sequence analyses,and cloned genes.We used prokaryotic expression systems to express odorant binding proteins(OBPs)and chemosensory proteins(CSPs),and also a combination of fluorescence competition and immunofluorescence localization.The main research results are described as follows:1.Insect olfactory proteins are located in the olfactory sensilla of antenna and the mouthparts.Observations of ultrastructure of these sensilla on the antennae of grape phylloxera showed:two trichoid sensilla are located on the scape,another two and a campaniformia sensillum are found on the pedicel.Seven trichoid sensilla and a primary rhinarium are located on the flagellum.Five trichoid sensilla are located on the tip of the processus terminalis and two are found at the near end.The primary rhinarium consists of a large placodea sensillum and four coeloconica sensilla.Three trichoid sensilla are found on the mouthparts of grape phylloxera,and are on each side of the upper surface of the junction of the labrum and anteclypeus;another one is on the front upper surface of the labrum.Some trichoid sensilla,and six pairs of basiconica sensilla are symmetrically located at the apex of the fourth labial segment.Two smooth mandibular stylets with food and salivary cannals are on in the inside surface,and a very short common duct is formed by the fusion at the end.2.A total of 27 olfactory proteins were identified by transcriptome analysis in grape phylloxera,and there were seven OBPs,seven CSPs,four ORs,one SNMP,and eight GSTs.DviOBPs have six conserved cysteine residues,which form three pairs of disulfide bonds,which are classic OBPs and plus-C OBPs.DviCSPs have four conserved cysteine residues forming two pairs of the disulfide bonds that are essential for the protein's spatial structure.DviORco and DviOR12 have seven transmembrane regions,and DviOR19 and DviOR43have six.DviSNMP1 has one transmembrane domain at both the N-terminal and C-terminals.Eight DviGSTs include seven cytosolic GSTs and one microsomal GST.These seven cytosolic GSTs are:DviGSTd1,DviGSTd2,DviGSTt1,DviGSTol,DviGSTs1,DviGSTs2and DviGSTs3.These are found in four different subfamilies:Omega,Delta,Theta,and Sigma.One microsomal GST,DviGSTm,has three transmembrane regions and a conserved sequence.Phylogenetic analyses showed that most of 27 olfactory proteins were closely related to these protein families of other insects.Semiquantitative polymerase chain reaction(sRT-PCR)was used to examine the tissue expressions of these transcripts,and the results revealed that DviOBP1,DviOBP6,DviOBP7,and DviSNMP1 are uniquely or primarily expressed in the head and not in the body.DviOBP2,DviGSTo1,and DviGSTs1 are more abundantly expressed in the body than in the head,and the other transcripts are abundantly expressed in both the head and the body.3.Gas chromatography-mass spectrometry(GC-MS)was used to identify the host plant volatiles of Kyoho grape vine roots after solid phase microextraction(SPME),and the results showed that these host plant volatiles were made up of 51 substances containing aldehydes(5kinds),alkenes(14 kinds),alcohols(12 kinds),alkanes(11 kinds),esters(5 kinds),and other(4 kinds).4.We successfully constructed recombinant expressions of the plasmids pET-28a(+)/DviOBP1,pET-28a(+)/DviOBP3,pET-28a(+)/DviOBP6,pET-28a(+)/DviCSP2,and pET-28a(+)/DviCSP5 from plasmid extractions,enzyme digestion,connection,and transformation methods.The expression vectors were induced by IPTG and proteins were purified using a Ni column.Finally,the soluble target proteins of DviCSP2 and DviCSP5were successfully obtained.The concentrations of the target proteins after purification were determined by the Bradford method.5.The binding affinities of DviCSP2,DviCSP5,and 19 host plant volatiles were measured by fluorescence ligand-binding assays and the results showed that two recombinant DviCSPs can combine with some host plant volatiles and both are combined with ester substances.DviCSP2 can combine with methyl linoleate,dibutyl phthalate,eucalyptol and camphene with K_i values of 168.40,222.19,351.94 and 349.00?M.DviCSP5 can combine with methyl linoleate,dibutyl phthalate,eucalyptol,camphene,hexanol,linalool,2-Ethyl-1-hexanol,and benzyl alcohol with K_i values of 27.36,21.57,22.62,70.04,91.06,14.44,72.52,and 81.72?M.DviCSP2 and DviCSP5 play an important role in helping grape phylloxera recognize host plant volatiles,especially esters.Furthermore,DviCSP5 has a stronger binding affinity.6.The purified DviCSP5 protein was used to successfully induce a polyclonal antibody in immune new Zealand white rabbits.The DviCSP5 was localized in the overall grape phylloxera organizations using immunofluorescence localization,and the results show that the antennae sensilla have no obvious fluorescent tags.The trichoid sensilla in the third and fourth segments of the mouthparts were marked.The distal portion of the middle foot,but not sensilla,was obviously fluorescent.In conclusion,DviCSP5 is mainly distributed in the sensilla of mouthparts and lymphatic fluid of the distal portion of the middle foot,but is not found in the antennae sensilla.