| The cotton bollworm,Helicoverpa armigera(Hubner),has developed resistance to multiple insecticides,among which,pyrethroids resistance was the most severe one.Increased metabolism of insecticides mediated by cytochrome P450s was an important mechanism for pyrethroids resistance.Many studies in China and other countries showed that several pyrethroids resistance-related P450 genes were over-expressed in resistant populations of H.armigera.The over-expression of P450 genes were mainly regulated by transcription procedure.In this study,the relative expression level of CYP6B7 and other pyrethroid resistance-related P450 genes in HDS population and field collected populations were investigated.The 5' untranslated region(5'UTR)of CYP6B7 was cloned,and the cis-acting element of 2-tridecanone and the responsive region of fenvalerate in the 5'UTR of CYP6B7 were identified.Besides,the differentially expressed genes in metabolism procedure and transcription factors between fenvalerate resistant and susceptible populations of H.armigera were investigated by RNA-Seq integrally.The study was aimed to elucidate the regulation mechanisms of the over-expression of CYP6B7.The results were listed below.1.Determination of resistance level and expression level of resistance-related P450 genes in field collected populations of H.armigeraSeveral populations of H.armigera were collected from Beijing,Hebei,Henan and Shandong province and the resistance level to some common used insecticides were determined.Compared with HDS population,the field collected populations showed high resistance ratio to fenvalerate,which were ranged from 5.4-to 114.7-fold,and low resistance ratio to lambda-cyhalothrin,phoxim and methomyl,with resistance ratio less than 10-fold.The synergism ratio to fenvalerate by PBO was the highest,ranging from 91.5 to 4497.2-fold,while DEF and DEM showed low synergism ratio to fenvalerate.The relative expression level of multiple resistance-related P450 genes in the sixth instar larvae were determined by real-time quantitative PCR.CYP6B7,CYP332A1 and CYP9A12 showed high over-expression level in the field-collected fenvalerate-resistant populations.2.Cloning and Characterization of the 5' UTR of CYP6B7Gene specific primers were designed according to the coding sequence of CYP6B7.A sequence of 1934 bp in length ahead of the start codon(ATG)of CYP6B7 5'UTR was cloned from HDS population by genome walking technique(GenBank accession no.KY196470).Blast search in NCBI indicated that the 5' UTR of CYP6B7 was similar with the complete sequence of CYP6B2,CYP6B6 and CYP6B7 of H.armigera and partial sequence of alpha subunit gene of voltage-gated sodium channel.The transcription start site was predicted as an A by the online software.Several transcription factor binding sites were predicted,including EcRE,XRE-AhR,Oct-1,Dfd,ftz,Eve and ZEN.The 5' UTR of CYP6B7 in Beijing population of H.armigera was also cloned ahead of ATG,which was 1395 bp in length.Alignment of the 5' UTR of CYP6B7 between HDS and Beijing populations revealed several nucleotides difference,including a short fragment deletion(732-791 bp)in HDS population and a long fragment deletion(961-1013 bp)in Beijing population.3.Induction of CYP6B7 by 2-tridecanone and the identification of cis-acting elementThe expression of CYP6B7 in midgut of H.armigera could be significantly induced by alleochemical 2-tridecanone.Stepwise deletion fragments of CYP6B7 5' UTR and promoter-less vector of pGL3-Basic were ligated,and CYP6B7-pGL3 plasmids(pGL3-(-1703/+232),pGL3-(-680/+232),pGL3-(-320/+232),pGL3-(-238/+232)and pGL3-(-72/+232))were constructed.The recombinant plasmids were transfected to Sf9 cell and the dual-luciferase activity were assayed.The results indicated that 2-tridecanone had significant induction effect(3.56-fold)on pGL3-(-320/+232),while had no induction effect on pGL3-(-238/+232).Two more constructs pGL3-(-292/+232)and pGL3-(-256/+232)between constructs pGL3-(-320/+232)and pGL3-(-238/+232)were generated by stepwise deletion.The results showed that 2-tridecanone had significant induction effect(2.15-fold)on pGL3-(-292/+232),while no induction effect on pGL3-(-256+232).M1,M2 and M3 were divided from sequence between-292 and-257 bp.Nucleotides mutation in M1 had no influence on the induction effect by 2-tridecanone,while mutation in M2 and M3 decreased the luciferase activity of pGL3-(-320/+232)significantly after 2-tridecanone treatment.The results suggested that the cis-acting element mediating the over expression of CYP6B7 was distributed in M3,which was between-280 and-257 bp in the 5'UTR of CYP6B7.4.Induction of CYP6B7 by fenvalerate and the identification of responsive regionThe expression of CYP6B7 in midgut of H.armigera could be significantly induced by artificial diets containing 0.1 mg/g fenvalerate after 48 h treatment.Induction to the five recombinant plasmids indicated that fenvalerate had significant induction effect(1.91-fold)on pGL3-(-320/+232).Further induction to the recombinant plasmids pGL3-(-320/+232),pGL3-(-292/+232),pGL3-(-256/+232)and pGL3-(-238/+232)showed that fenvalerate had significant induction effect(1.75-fold)on pGL3-(-292/+232),while no induction effect on pGL3-(-256/+232);However,the basal and fenvalerate-induced relative luciferase activity of pGL3-(-292/+232)was significantly lower than that of pGL3-(-320/+232).The results suggested that the responsive region of fenvalerate was between-320 and-257 bp of 5'UTR of CYP6B7.Compared with the basal relative luciferase activity of construct pGL3-(-292/+232),the combination induction by 2-tridecanone and fenvalerate was 2.03-fold,while 2.52-and 1.59-fold by 2-tridecanone and fenvalerate,respectively.5.RNA-Seq analysis of the fenvalerate resistant and susceptible populations of H.armigeraA fenvalerate resistant population(BJR)with more than 1275-fold resistance ratio was obtained by series selection by fevalerate from Beijing population.RNA-Seq analysis identified 6130 unigenes up-regulated in BJR population compared with that in HDS population.33 metabolism procedure related unigenes were screened and further validated by RT-qPCR.The results indicated that there were 17 unigenes significantly up-regulated,including 10 P450,1 EST,1 GST and 5 UGT unigenes.Analysis on the transcription factors revealed 199 up-regulated and 158 down-regulated unigenes.Maf and Arh nuclear translocator,which were reported to be the transcription factor of P450 genes and belonged to TF_bZIP and bHLH transcription factor families,respectively,were found among the up-regulated unigenes. |
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