Identification And Application Of Odorant Binding Proteins And Semiochemicals In Carpomya Vesuviana Costa

In order to study olfactory mechanism of odorant binding proteins in Carpomya vesuviana Costa,we have firstly identified the olfactory proteins.According to solid phase microextraction(SPME)and gas-chromatography/massspectrometry(GC/MS),the volatiles of adults and host plants were collected and identified.Next,combining the assays of Electroantennogram(EAG),the volatiles with EAG reactivity were screened.After expression and purification of OBPs,the compounds having EAG reactivity were studied of the binding affinity.Finally,the molecular model of OBPs was constructed.The results are as followings:The transcriptome sequence results showed that there were 64,662unigenes with total length 57,187,525 and mean length 884 bp.There were 29298 unigenes(about 45%of total unigenes)could be annotated to different GO classification,including 10382 unigenes which were annotated to biological process,9516 to cellular components,and 9400 unigenges annotated to molecular function.We have identified and annotated for the first time 15 ORs and 22 GRs,2 SNMPs and 2CSPs.Six complete sequences encoding OBPs were obtained.The predicted amino acid sequences of CvesOBP1,3,4,and 6 revealed that they belonged to the "classic" OBPs family,while CvesOBP2 and CvesOBP5 belonged to the "Minus-C" family.SPME and GC/MS were used to collect and identify volatiles.In audlts,one volatile compound,nonanal,have firstly identified,which could intrigue both sexual adults EAG response.In the host plants,chamigrene,caryophyllene,camphene,(Z)-3-Hexen-l-ol acetate andocimene were collected and identified.Additionally,geranyl acetate and ?-farnesene which were regard as pheromones in Ceratitis capitata,could also induce EAG response in male and female adults.The trap catches in field showed that six semiochemicals are better than yellow sticky board,and the nonanal has the best effect.SqRT-PCR reveals that CvesOBP1-6 could highly express in abdomen,head and thorax.CvesOBP1 and CvesOBP2 could also express in wing,and the expression of CvesOBP1,CvesOBP5 and CvesOBP6 were also find in leg.Six OBPs were expressed in prokaryotic expression system and purified,and two of them were found as inclusion body protein.Then the affinity of the six CvesOBPs to various compounds were measured.Ligand binding experiments indicated that CvesOBP2 had affinity only to(Z)-3-hexenhexen-l-ol acetate,while OBP5 and OBP6 exhibited a broad spectrum binding affinity to compounds with relatively low molecular weights.OBP1 and OBP4 showed some affinity to caryophyllene and chamigrene.Additionally,OBP3 showed relatively high binding affinity to a-famesene,and a low affinity to geranyl.In the study of three-dimensional structure,only OBP3 has well-matched model to construct three-dimensional structure.According to assessment of it,there were only five amino acid with unreasonable conformation,and the 66th phenylalanine has the most effect on stability of protein.This structure exist six compact a-helix and lumen with combining capability.It is also showed that hydrophilic amino acids were found out of the lumen,while hydrophobic amino acids in the lumen.