Identication And Functional Research On Odorant Binding Proteins From Liriomyza Sativae Blanchard

The leafminer,Liriomyza sativae Blanchard,is a polyphagous insect specie seriously damaging to vegetables and ornamental plants.In order to study the function of odorant binding proteins of L.sativae,twenty odorant binding proteins genes(OBPs)with full-length cDNA coding region from L.sativae adult transcriptome were identified by using RACE-PCR amplification.The main conclusions are as follws:1.The twenty encoding putative odorant binding proteins were named as LsatOBP1-15 and LsatPBPRP2-6,corresponding to 9 Classic,5 Minus-C,4 Plus-C and 2 Dimer subfamily OBPs;The relative expression of all LsatOBPs at stages of larvae,pupae and adults were measured by Real-time quantitative PCR(RT-qPCR).The results showed that 4 LsatOBPs highly expressed in the larvae stage,Two LsatOBPs were expressed highly in pupae stage.And the rest of 14 LsatObps were highly expressed in adults.The relative expression of 14 LsatOBPs at different ages in male and female adults are differnece with each other,8 of those 14 LsatOBPs gene expression show no significant difference in newly emerged male and female adults,all those 14 LsatOBPs showed significantly higher in male than in female adults,whereas 5 of those 14 LsatOBPs shows highly expressed in male at the three day age than female adults at the same age.Those differences in expression of LsatOBPs may be related to the male behavior.2.Seven kinds of recombinant LsatOBPs proteins purified from prokaryotic expression were obtained to found the binding capacity with 25 kinds of selected odorants chemical ligands were measured by use of N-phenyl-naphthylamine(1-NPN)as a fluorenscent probe.The results indicated that all 7 LsatOBPs could specific bind with odor compent β-Ionone.LsatOBP13,LsatPBPRP2 and LsatPBPRP6 could bind with the general green leaf volatile 1-Octen-3-ol;LsatOBP1,LsatPBPRP4 and LsatPBPRP6 could strongly bind with the limonene which a key compouds associated with tomato,the preference host plant of L.sativae.1-Octen-3-ol and limonene could cause distinct EAG responses and behavioral responses in L.sativae in research papers.We suggested that specific volatiles recognized by LsatOBPs relative to behavior of insect.The difference identification ability of LsatOBPs were assiociated to the function of LsatOBPs.LsatOBP1 could strongly bind to fifteen kinds of selected odor compents,mainly showed strong ability bind to many kinds of fatty acids in plants;LsatPBPRP6 was also compatible with fifteen kinds of selected odorants,but most of those selcected odorants were the green leaf odors of plants.All results indicate that different LsatOBPs might recognize different odorous substances,and LsatOBPs might not only play role in the process of olfactory recognition.All seven kinds of LsatOBPs couldn’t bind with sex pheromone of house fly,Musca domestica,showed LsatOBPs have specific identification to pheromone.The study on binding properties of LsatOBPs might have value in understanding the mechanism of the olfactory recognition of Liriomyza and seeking and developing new attractants and repellents.3.The distribution of LsatPBPRPs2-6,LsatOBP1 和 LsatOBP13 in the sensillas of antenna and proboscis of L.sativae were detected by co-immunofluorescence localization.There were Trichoid senailla I,Trichoid senailla II,Basiconic sensilla(BS),Clavate sensilla(CS)and Grooved sensilla(GS)and olfactory pit on the funicular of L.sativae antenna.The distribution of LsatOBPs in different olfactory sensilla may be related to the function of olfactory sensilla.The results showed that LsatPBPRP3 and LsatPBPRP6 were co-expressed in Trichoid sensilla II(TSII),Basiconic sensilla(BS),Clavate sensilla(CS)and Grooved sensilla(GS);LsatPBPRP3 co-expression with LsatPBPRP4 in Basiconic sensilla;LsatPBPRP2 co-expression with LsatPBPRP3 in Trichoid sensillaII and Basiconic sensilla;LsatOBP1 and LsatPBPRP6 could co-expression in Grooved sensilla;LsatPBPRP5 could express in Trichoid sensilla I;LsatPBPRP4 and LsatOBP13 were co-expressed in olfactory pit of L.sativae antenna;LsatPBPRP2,3,6 and LsatOBP1,13 were largely expressed in the Proboscis of L.sativae.According to the results of the distribution of LsatOBPs and the binding characteristics of LsatOBPs,we speculated that LsatPBPRP4 and LsatOBP13 which could express in olfactory pit might were the important odorant binding proteins in the olfactoru recognition process of L.sativae.