| Aphis gossypii Glover(Hemiptera:Aphididae)can damage a variety of agricultural crops,so it is very important for cotton aphids to evolve adaptive mechanisms to various allelochemicals from host plants.Cytochrome P450 monooxygenases(P450s)are known to be involved in the metabolism or detoxification of plant allelochemicals in insects.Our studies are based on Illumina sequencing platform?molecular biology study and bio-informatics analysis,aim to provide a fundamental and rich resource for exploring aphid functional genes in A.gossypii.This provides further research targets aimed at understanding the regulatory mechanisms of P450 genes expression and their interactions with plant allelochemicals in insect pests.The main results are as follows:1.A transcriptome data set and five expression profile data sets of A.gossypii samples were analyzed by Illumina sequencing platform.In total,53,763,866 reads were assembled into 1,963,516 contigs and 28,555 unigenes.Compared with the control,619 genes were significantly up-or down-regulated in the treatment group by 2-tridecanone.There were 516,509,and 717 of differential expression genes in tannic acid,quercetin,and gossypol treatment groups,respectively.Furthermore,there were 4 of 54 putative cytochrome P450 genes and 1 of 7 putative carboxylesterases down-regulated in all treatment groups by four plant allelochemicals.When aphids fed on 2-tridecanone,tannic acid,and quercetin,only one P450 gene was up-regulated.These results show that plant allelochemical stress can induce differential gene expression in A.gossypii.2.The expression of P450 genes in insects can be induced by plant allelochemicals.To understand the induction mechanisms,we measured the expression profiles of five P450 genes and their promoter activities under the induction of plant allelochemicals.The results showed that the inducible expression of CYP6CY19 was the highest among three CYP6 subfamily genes,followed by those of CYP6CY22 and CYP6DA1.All these four plant allelochemicals(2-tridecanone,gossypol,tannic acid and quercetin)were able to induce the expression of these three P450 genes.Tannic acid had a better inductive effect than other three plant allelochemicals.The transcript level of CYP305A1 were significantly induced by two plant allelochemicals,gossypol and 2-tridecanone.And the transcript levels of CYP4CJ1 were significantly induced by both gossypol and tannic acid.3.To further explore the molecular genetics mechanism behind the inducible expression of these P450s,In this study,a full-length five P450 gene were cloned and their promoters were identified from A.gossypii.Transient transfection results showed that the regions from-687 to +586 bp of CYP6DA1,from-666 to +140 bp of CYP6CY19 and from-530 to +218 bp of CYP6CY22 were essential for basal transcriptional activity.The cis-elements for plant allelochemicals induction were identified between-193 and +56 bp of CYP6DA1,between-157 and +140 bp of CYP6CY19 and between-108 and +218 bp of CYP6CY22.These promoter regions were found to contain a potential aryl hydrocarbon receptor element binding site with a conservative sequence motif 5'-C/TAC/ANCA/CA-3'.Furthermore,the 5'-end promoter region from-810 to-581 bp of CYP305A1 was shown as an essential plant allelochemical responsive region and had a cis-element 5'-CACACTA-3' of aryl hydrocarbon receptor(AhR)binding site.And a region in the 5'untranslated region of CYP4CJ1(from-1173 to-1422)was identified,which could be an essential plant allelochemical responsive region.In addition,there was an 1094 bp overlapping region between CYP305A1 promoter and the venom protease gene.When the expression of CYP305A1 was knocked down by RNAi with the CYP305A1 dsRNA,the expression of the venom protease gene was decreased.However,the knock-down of the expression of the venom protease gene did not affect the CYP305A1 expression.These results are important for understanding the roles of P450 genes in the plant allelochemical resistance of A.gossypii,and the regulatory mechanism of the P450 gene expressions by other genes or factors.These results are important for understanding the roles of P450s in the plant allelochemical resistance of A.gossypii,and are meaningful for understanding the regulatory mechanism of P450 genes.4.In this study,we used yeast one-hybrid to screen 12 potential transcription regulators of the CYP4CJ1 from A.gossypii that respond to the plant secondary toxicant,which were named Prey1 to Prey 12,respectively.According to the NCBI database blast,Prey3?Prey 4?Prey 5?Prey 6?Prey 8 were same sequences and were 18s RNA with Prey 1;Prey 11 was aphid symbionts;Prey 12 was 16s RNA.Prey 7 and Prey 9 were same sequences.Prey 2 may be potential larval cuticle protein of four species aphids(Myzzus persicae?Acyrthosiphon pisum?Melanaphis sacchari and Diuraphis noxia);Prey7 were four kinds of AFLP fragments of Daktulosphaira vitifoliae;Prey 10 were two uncharacterized proteins of Diaphorina citri.Some studies had been showed that insect larval cuticle protein was involved in resistance processes,Type RR-2 of CPR family from aphids was important for pest resistance to insecticides.This is consistent with the results of this study.Our results showed that Prey2 belongs to CPR family and may participates in detoxification metabolism of plant allelochemicals with P450s. |
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