Antennal Transcriptome Analysis And Bioinformatics Analysis Of Olfactory Genes In The Drosicha Corpulenta

Drosicha corpulenta Kuwana(Hemiptera:Cocoidea:Monophlebidae),is a variety of phytophagous insect which has wide ranges of host,whose nymph and female imago gather at branch and burgeon feeds the juice by the way of piercing-sucking.When the density is too much,it often makes the sprout dead.Due to the maskant that composed by waxiness from D.corpulenta's surface,it is hard to control by low efficiency of manual clear and chemical prevention.Besides,too much using of chemical insecticide is not only killing the natural enemy but also contaminate the fruit and environment.Therefore,this essay researches the albumen in D.corpulenta's antenna which play roles in the sense of olfactory process,from the view of the effect of insect chemical communication to pest control,and gives a new angle in studying olfactory genes function to D.corpulenta and provides the theoretical basis in biological protection.The research content includes(1)The article present the first antennal transcriptome sequencing information for the adult female D.corpulenta by Illumina HiseqTM 2000,then assembly,annotation and homology analysis;forecasting D.corpulenta's olfactory genes and concluding information of these genes;compare the classic OBPs and CSPs sequence of the D.corpulenta respectively,then using the former result build phylogenetic trees respective.(2)Basing on the antennal transcriptome sequencing information and analysis result of female adult D.corpulenta we gain the DcorOBP4,5,6,7,8,14 and DcorCSP2's coding sequences by way of RT-PCR cloning technology;analyzing these amino acid sequences information by bioinformatics methods;building these proteins three-dimensional structure.Result as follows:1.We present the first antennal transcriptome sequencing information for the female D.corpulenta and identify the major olfactory genes.A total of 33,741,845 clean reads were obtained,from which 16,334 unigenes were assembled.Gene ontology(GO)and Non-supervised Orthologous Groups(eggNOG)analyses were used for functional classification.Compared with other species,the unigenes in D.corpulenta's antenna are similar to Acyrthosiphum pisim Harris most to 18.31%.2.We identified 16 transcripts encoding putative odorant-binding proteins(OBPs),2 chemosensory proteins(CSPs),1 sensory neuron membrane proteins(SNMPs),21 odorant receptors(ORs)and 7 gustatory receptors(GRs).Predicted protein sequences were mainly compared with Aphididae,Miridae and Phenacoccus solenopsis Tinsley(only CSPs genes were found in NCBI database).The results of phylogenetic trees showed that eight OBPs of D.corpulenta were clustered with Aphididae and some clusters included only OBPs from D.corpulenta.Both of two CSPs were grouped with P.solenopsis.The genes reported here present new insight to the role of identified olfactory genes in scale insect,provide possible ideas for insect pest control and improve currently biological control strategies.3.Basing on the antennal anscriptome sequencing information and analysis result of female adult D.corpulenta,we get the coding sequence of the OBPs(DcorOBP4,5,6,7,8,14)and DcorCSP2's coding sequences by way of RT-PCR cloning technology,and prediction analysis these amino acid sequences on physicochemical property,signal peptide and hydrophilic-hydrophobic property in the way of bioinformatics methods.The analysis revealed that all of these proteins have complete open reading frame(ORF),there are 19?29 amino acid's signal peptide on N-terminal,the isoelectric point is between 4.12-9.90,the relative molecular mass is 15.6-17.0 KDa,they all are hydrophilic proteins.The OBPs(DcorOBP4,5,6,7,8,14)have 6 conserved cysteine sites,which is conform to typical odorant binding protein family feature.There are 4 conserved cysteine sites in DcorCSP2,it is also conform typical Chemosensory protein family characteristic.Using SWIISS-MODEL homologous modeling online server build the 3D-structure of OBPs(DcorOBP4,5,6,7,8,14)and DcorCSP2.It's supplies strong basis in researching these protein's role to identify odorant compound like host and non-host volatiles and sex pheromone.The research manifests that 6 conserved cysteine sites of OBPs(DcorOBP4,6,7,8)form 3 pairs of disulfide briages,and 2 pairs by DcorCSP2 both of which play a role in protein structure to make it stabilize.There are only 2 pairs of disulfide bond,and 2 conserved cysteine sites are not connected in DcorOBP5 and DcorOBP14,it might relate to the template used for modeling.These odorant binding protein's(DcorOBP4,5,6,7,8,14)5 alpha helix form to a hydrophobic pocket which alpha three is at one side of the pocket but not participate in hydrophobic pocket formation.different with that,In the 3D-structure of the odorant binding protein Hob1OBP1 and Hob1OBP2 of the scarab bettle Holotrichaia oblita Fald,and LimgOBP1 of the Locusta migratoria manilensis Meyen,5alpha helix(?1,?2,?4,?5,?6)form to a hydrophobic pocket,which alpha two is not participating in hydrophobic pocket formation.In the 3d-structure of BmorPBP of the Bombyx mori Linnaeus,alpha two and alpha three are not participate in hydrophobic pocket formation.Therefore,we speculate that there is some discrepancy in forming hydrophobic pocket by OBP's 3D-structure from different insect.