| Insects recognize these complex molecules mainly through their sensitive olfactory systems,and chemical reception often triggers corresponding behavioral responses,such as mating,searching for hosts,selecting oviposition sites and so on.There are several types of proteins involved in odor detection,including odorant receptors(ORs),ionotropic receptors(IRs),odorant binding proteins(OBPs),chemosensory proteins(CSPs)and sensory neuron membrane proteins(SNMPs).Therefore,the study of olfactory genes is helpful for understanding the molecular mechanism of insect olfactory recognition.Bactrocera minax(Enderlein)is a devastating pest that harms citrus fruits in China,India and other countries.This insect pest is also an important quarantine pest in the world.To understand the olfactory system of this economically important species,we analyzed B.minax transcriptomes from the antennae and legs.We analyzed the expression patterns of olfactory genes in antennae and legs,and then combined with the results of evolutionary tree analysis,we selected four odorant receptor genes were selected for functional study.The main results are as follows:1.Transcriptomes analysis and olfactory genes identification of B.minax.The 12 c DNA libraries from B.minax were sequenced using the Illumina Hi Seq 2500 platform.73.60 Mb,73.60 Mb,73.96 Mb,74.07 Mb,71.57 Mb,74.24 Mb,74.30 Mb,74.70 Mb,74.43 Mb,73.97 Mb,73.46 Mb and 74.61 Mb clean reads were generated for the 12 libraries,respectively.Assemblies led to the generation of 42,281,38,540,41,909,42,019,41,584,43,952,35,953,36,572,34,810,33,967,32,538 and 32,622 unigenes from the 12 libraries,respectively.After merging and clustering,72,013 all-unigenes were obtained.The total length,average length,N50 and GC content of all-unigenes are 124,877,089 bp,1,734 bp,3,855 bp and 37.43%,respectively.We identified a total of 147 chemosensory genes in the B.minax antenna and leg transcriptomes,including 59 Bmin ORs,16 Bmin IRs,25 Bmin GRs,39 Bmin OBPs,five Bmin CSPs and three Bmin SNMPs.2.Expression pattern analysis of olfactory genes in antennae and legs of B.minax.The FPKM values were used to evaluate the expression levels of all chemosensory genes(Bmin ORs,Bmin IRs,Bmin OBPs,Bmin CSPs and Bmin SNMPs)in the female and male antennae and in female and male legs of B.minax.Among the Bmin ORs genes,most genes were highly expressed in male and female antennae,of which Bmin Orco was the highest expressed,and Bmin OR2 was also expressed in male and female legs.Among the Bmin IRs genes,9 Bmin IRs(Bmin IR93 a,Bmin IR25 a,Bmin IR76 a,Bmin IR84 a,Bmin IR76 b,Bmin IR75 d,Bmin IR8 a,Bmin IR41 a and Bmin IR92 a.4)were highly expressed in the male and female antennae,of which 3 Bmin IRs(Bmin IR93 a,Bmin IR25 a and Bmin IR75d) genes were also expressed in the male and female legs.Among the Bmin OBPs genes,most of the Bmin OBPs genes are highly expressed in male and female antennae,of which 13 Bmin OBPs(Bmin OBP27,Bmin OBP33,Bmin OBP7,Bmin OBP20,Bmin OBP5,Bmin OBP24,Bmin OBP16,Bmin OBP21,Bmin OBP13,Bmin OBP3,Bmin OBP35,Bmin OBP2 and Bmin OBP12)genes are also highly expressed in male and female legs.Among the Bmin CSPs genes,Bmin CSP3 and Bmin CSP2 are highly expressed in the male and female antennae,and Bmin CSP1 is not only highly expressed in the male and female antennae,but also highly expressed in the male and female legs.In the Bmin SNMPs genes,Bmin SNMP1 and Bmin SNMP3 are highly expressed in male and female antennae.A significant analysis of the differential expression of all olfactory genes(Bmin ORs,Bmin IRs,Bmin OBPs,Bmin CSPs and Bmin SNMPs)in male and female antennae showed that none of the olfactory genes had significant differential expression in male and female antennae.We used the semi-quantitative reverse transcription PCR(RT-PCR)to analyze the expression patterns of the candidate Bmin ORs in the antennae and legs of both sexes.All Bmin ORs genes were expressed in the female and male antennae,of which 4 Bmin ORs(Bmin OR26,Bmin OR49,Bmin OR50 and Bmin OR55)genes were also expressed in male and female feet,and 7 Bmin ORs(Bmin Orco,Bmin OR12, Bmin OR14,Bmin OR16,Bmin OR24,Bmin OR45 and Bmin OR58)genes were highly expressed in male and female antennae,and there is no difference in expression between male and female.And 7 Bmin ORs(Bmin OR5,Bmin OR6,Bmin OR10,Bmin OR26,Bmin OR34,Bmin OR50 and Bmin OR57)were more expressed in the male antennae than in the female antennae.In addition,the expression of Bmin OR49 in male and female legs was higher than that in male and female antennae.3.Clone and functional characterization of odorant receptor in B.minax.In this study,four ordinary ORs(Bmin OR3,Bmin OR12,and Bmin OR16 and Bmin OR24)were identified and cloned using data generated from transcriptomic analyses.RT-PCR analysis of Bmin OR expression showed that Bmin OR3,Bmin OR12,Bmin OR16 and Bmin OR24 were expressed in the antennae,since their expression was not detected in the head,thorax,abdomen,and leg tissues.The expression of all four Bmin ORs shows no difference between male and female.Among these,the expression levels of Bmin OR12,Bmin OR16 and Bmin OR24 are significantly higher than that of Bmin OR3 in both the male and female antennae.Bmin OR3,Bmin OR12,and Bmin OR16 were co-expressed with the Bmin Orco in Xenopus oocyte,and functionally characterized using the voltage clamp recording system.Our results showed that the oocytes expressed in the three Bmin ORs are activated by at least one of the plant volatiles.Bmin OR3/Bmin Orco only has strong responses to 1-octen-3-ol.Bmin OR12/Bmin Orco responded to eight compounds: benzyl alcohol,(S)-(+)-carvone,1-octanol,butyl propionate,butyl acrylate,(Z)-3-hexenyl acetate,benzaldehyde,and methyl salicylate.Bmin OR16/Bmin Orco only responded weakly to undecanol.Bmin OR24/Bmin Orco specifically responds to stimulation by plant volatiles linalool and methyl eugenol.In behavioral experiments,the females has no behavioral effect on 1-octen-3-ol at a concentration of 1 ?g/?L. |
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