Identification of the 2-tridecanone cis-acting element in the promoter of cytochrome P450 CYP6B7 in Helicoverpa armigera

The expression level of cytochrome P450 genes in insects can be induced by plant allelochemicals,which is important for insects to adapt to host plants.Cytochrome P450CYP6B 7has been reported to be involved in pyethroid insecticide resistance in Heli- coverpa armigera,and its transcription level was induced by some inducers.Currently,the regulatory mechanism of the induced expression of CYP6B7remains unknown,although it is very important for understanding the detoxification mechanism to allelochemicals in host plants.The objective of the present study was to investigate the eis-acting ele- ment in the promoter of CYP6B7 mediating the inducible up-regulation of CYP6B7in H.armigera by 2-tridecanone.The promoter region of CYP6B7was cloned by genome walking technique and analyzed by transient transfeetion assay.Progressive 5'deletion of the promoter region of CYP6B7revealed that the relative luciferase activity of construct -320/+232could be significantly induced by 2-trideeanone.Further stepwise deletion between -320 and -238 bp found that construct -292/+232 could also be significantly induced by 2-tridecanone,but the adjacent construct -256/+232could not,suggesting the essential role of the sequence between -292 and --257 bp for 2-tridecanone induction. Nucleotide mutations between -292 and -281 bp had no influence on the induction ef- fect by 2-tridecanone,but nucleotide mutations between -280 and -257 bp significantly decreased the induction effect.These results demonstrated that the cis-acting element for 2-trideeanone induction was between -280 and -257 bp in the promoter of CYP6B7.

Alcohol dehydrogenase 5 of Helicoverpa armigera interacts with the CYP6B6 promoter in response to 2-tridecanone

Alcohol dehydrogenase 5(ADH5)is a member of medium-chain dehydro-genase/reductase family and takes part in cellular formaldehyde and S-nitrosoglutathione metabolic network.2-tridecanone(2-TD)is a toxic compound in many Solanaceae crops to defend against a variety of herbivory insects.In the broader context of insect development and pest control strategies,this study investigates how a new ADHS from Helicoverpa armigera(HaADH5)regulates the expression of CYP6B6,a gene involved in molting and metamorphosis,in response to 2-TD treatment.Cloning of the HaADH5 complementary DNA sequence revealed that its 1002 bp open reading frame encodes 334 amino acids with a predicted molecular weight of 36.5 kD.HaADH5 protein was purified in the Es-cherichia coli Transetta(pET32a-HaADH5)strain using a prokaryotic expression system.The ability of HaADH5 protein to interact with the 2-TD responsive region within the promoter of CYP6B6 was confirmed by an in vitro electrophoretic mobility shift assay and transcription activity validation in yeast.Finally,the expression levels of both HaADH5 and CYP6B6 were found to be significantly decreased in the midgut of 6th instar larvae after 48 h of treatment with 10 mg/g 2-TD artificial diet.These results indicate that upon 2-TD treatment of cotton bollworm,HaADHS regulates the expression of CYP6B6 by interacting with its promoter.As HaADH5 regulation of CYP6B6 expression may con-tribute to the larval xenobiotic detoxification,molting and metamorphosis,HaADH5 is a.candidate target for controlling the growth and development of cotton bollworm.