| Insect olfaction is mainly accomplished by a large number of sensilla covering the surface of antennae.Volatile odorants penetrate the sensillum lymph through cuticular pores and are recognized and bound by odorant-binding proteins(OBPs).Then,OBPs transport odorants through the sensillum lymph to the olfactory receptors(ORs)embedded in the dendrites of olfactory receptor neurons(ORNs).Then,signals are transmitted to the central nervous system,and the insect makes corresponding behavioral responses.Once ORs are activated and chemical signals are conveyed,the signal molecules must be rapidly inactivated to terminate the continuous stimulation,enabling insects to detect the changes of chemical signals in their environments.At present,it has been demonstrated that odorant-degrading enzymes(ODEs)play important roles in inactivating the odorant molecules by degradation.Cytochrome P450(P450s or CYPs)is an important odorant degrading enzymes that play an important role in the olfactory behavior of insects.Locusta migratoria,as an important agricultural pest in the world,has caused great destruction to crops,especially to wheat,corn,sorghum and other gramineous crops,and led to great economic losses to agricultural production.ODEs play indispensable roles in locusts receiving continuously external information.In this paper,the characteristics of P450 in the antennae of L.migratoria were systematically studied by transcriptome,RT-q PCR and other methods.The physiological functions of P450 in the antennae of L.migratoria were studied by using RNA interference method combined with electroantennogram and behavioral responses.The results are as follows: 1?Construction and analysis of antennal transcriptome of L.migratoriaA total of 153,792 transcripts with 11.07 Gb of base sequences were obtained from the antennal transcriptome database of L.migratoria.After assembly,75,444 unigenes were obtained with an average length of 947 bp.Unigenes was annotated in different databases.Overall,41.12% unigenes were annotated.These data provide important information for the subsequent cloning,characterization and functional analysis of P450 genes.2?Searching and screening of P450 gene in L.migratoria antennaeA total of 92 P450 genes were searched from the antennal transcriptome database of L.migratoria.Among them,68 sequences were assembled into the full-length sequences and 24 sequences were partial-lengent fragments.Phylogenetic analysis was carried out based on the 68 full-length P450 sequences.The results showed that most of these 68 P450 s are distributed into CYP3 and CYP4 clans.It was speculated that these P450 genes might be involved in the degradation of exogenous substances.First,the distribution of the 68 full-length P450 genes in different tissues was analysed by RT-PCR.Fourty-six genes were expressed in the antennae of L.migratoria.Then,RT-q PCR was used to determine the expression of 46 genes screened in more tissues.Among them,4 genes(CYP4C99,CYP6FD5,CYP6NY1 and CYP3327A1)were antennae-specific and 8 genes(CYP6MU1,CYP305M1,CYP3117C1,CYP3117D1,CYP3117E1,CYP3117F2,CYP3114A1 and CYP3372A1)were antennae-rich.It suggested that these P450 genes might degrade odorants and played roles in the olfactory behavior of L.migratoria.3 ? Molecular characteristics of CYP6FD5 and CYP6MU1 genes in L. migratoriaCYP6FD5 and CYP6MU1 genes were selected to study their molecular characteristics.The full length c DNA sequences of the two genes were verified by RT-PCR amplification and sequencing.Then the gene sequences were transferred to Genebank(accession numbers: KY852428 and KY852414).Bioinformatics analysis showed that the two anino acid sequences have 5 conserved regions of P450.Phylogenetic analysis showed that the two P450 s were clustered closely to CYP6 a family in Drosophila melanogaster.Tissue expression patterns found that the two genes were highly expressed in the antennae.CYP6FD5 was specifically expressed in the antennae.CYP6MU1 was predominatly expressed in the antennae,followed by the maxillary palp and tarsus.Developmental stage expression patterns showed that the two genes were expressed in the whole developmental stage of antennae.These results provided a solid foundation for the future functional research.4?Functional analysis of CYP6FD5 and CYP6MU1 in the antennae of L.migratoriaCYP6FD5 and CYP6MU1 were significantly silenced after ds RNA of the two genes were injected into the antennal sockets of L.migratoria for 24 and 48 h.The electrophysiological and behavioral responses of the 4th-instar locusts to the volatiles of gramineae and locusts were measured by RNA interference combined with electroantennogram and behavior detection.The results showed that the EAG responses of the female L.migratoria to decanal,cis-3-hexenyl acetate,and trans-2-hexen-1-al was decreased while those of the male locusts to decanal,cis-3-Hexenylacetate,trans-2-Hexen-1-al,4-vinylanisole,benzyl cyanide and benzaldehyde were reduced after silencing of CYP6FD5 gene.After silencing of CYP6MU1 gene,the EAG responses of the female L.migratoria to nonanal,guaiacol,and 4-vinylanisole was decreased while those of the male locusts to trans-2-hexen-1-al,benzaldehyde,4-vinylanisole,and decanal were declined.The behavioral responses of the locusts to wheat had no significant changes after CYP6FD5 or CYP6MU1 were silenced.These results suggested that CYP6FD5 and CYP6MU1 played roles in the olfactory behavior of L.migratoria.In summary,4 antennae-specific and 8 antennae-rich P450 genes were screened from the L.migratoria antennae by antennal transcriptome construction and RT-q PCR investigation.The functions of CYP6FD5 and CYP6MU1 genes in the olfactory behavior of L.migratoria were analysed by RNA interference combined with electroantennogram and behavioral response.These results lay a solid foundation for the systematic and in-depth studies on the function and degradation mechanism of antennae P450 s in the olfactory behavior of L.migratoria in the future,and provide a theoretical basis for the green behavior-based prevention and control technology of locusts. |
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