Transcription factors play an important role in regulating the expression of detoxification genes (e.g. P450s) that confer insecticide resistance. Our previous study identified a series of candidate transcription factors (CYP6B7-fenvalerate association proteins, CAPs) that may be related to fenvalerate-induced expression of CYP6B7 in a field HDTJ strain of H. armigera. Whether these CAPs can mediate the transcript of CYP6B7 induced by fenvalerate in a susceptible HDS strain of H. armigera remains unknown. Further study showed that the expression levels of multiple CAPs were significantly induced by fenvalerate in HDS strain. Knockdown of CAP19 [fatty acid synthase-like (FAS)], CAP22 [polysaccharide biosynthesis domain-containing protein 1 (PBDC1)], CAP24 [5-formyltetrahydrofolate cycloligase (5-FCL)], CAP30 [peptidoglycan recognition protein LB-like (PGRP)] and CAP33 [NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11 (NDUFA11)] resulted in significant inhibition of CYP6B7 and some other P450 genes expression; meanwhile, the sensitivity of HDS strain larvae to fenvalerate was significantly increased. In addition, PBDC1, PGRP and NDUFA11, either alone or in combination, could significantly enhance the activity of CYP6B7 promoter in HDS strain, as well as the expression level of CYP6B7 gene in Sf9 cells line. These results suggested that PBDC1, PGRP and NDUFA11 may be involved in the transcript regulation of key detoxifying genes in response to fenvalerate in HDS strain of H. armigera.

As we known, inducibility is an important feature of P450 genes. Previous studies indicated that CYP6B7 could be induced and involved in fenvalerate detoxification in Helicoverpa armigera. However, the regulatory mechanism of CYP6B7 induced by fenvalerate is still unclear. In this study, CYP6B7 promoter of H. armigera was cloned and the cis-acting element of fenvalerate was identified to be located between -84 and - 55 bp of CYP6B7 promoter. Subsequently, 33 candidate transcription factors (CYP6B7-fenvalerate association proteins, CAPs) that may bind to the cis-acting element were screened and verified by yeast one-hybrid. Among them, the expression levels of several CAPs were significantly induced by fenvalerate. Knockdown of juvenile hormone-binding protein-like (JHBP), RNA polymerase II-associated protein 3 (RPAP3), fatty acid synthase-like (FAS) and peptidoglycan recognition protein LB-like (PGRP) resulted in significant expression inhibition of CYP6B7, and increased sensitivity of H. armigera to fenvalerate. Co-transfection of reporter gene p (-84/-55) with pFast-CAP showed that JHBP, RPAP3 and PGRP could significantly increase the activity of CYP6B7 promoter. These results suggested that trans-acting factors JHBP, RPAP3 and PGRP may bind with cis-acting elements to regulate the expression of CYP6B7 induced by fenvalerate, and play an important role in the detoxification of H. armigera to fenvalerate.

Cytochrome P450 CYP6B7 has previously been proved to be associated with fenvalerate-resistance in Helicoverpa armigera. Here, how CYP6B7 is regulated and involved in the resistance of H. armigera is studied. Seven base differences (M1-M7) were found in CYP6B7 promoter between a fenvalerate-resistant (HDTJFR) and a susceptible (HDTJ) strain of H. armigera. M1-M7 sites in HDTJFR were mutated into the corresponding base in HDTJ, and pGL3-CYP6B7 reporter genes with different mutation sites were constructed. Fenvalerate-induced activities of reporter genes mutated at M3, M4, and M7 sites were significantly reduced. Transcription factors Ubx and Br, whose binding sites contain M3 and M7, respectively, were overexpressed in HDTJFR. Knockdown of Ubx and Br results in significant expression inhibition of CYP6B7 and other resistance-related P450 genes, and increase of sensitivity of H. armigera to fenvalerate. These results indicate that Ubx and Br regulate the expression of CYP6B7 to mediate the fenvalerate-resistance in H. armigera.

Cytochrome P450-mediated detoxification plays an important role in the development of insecticide resistance. Previous studies have shown that cytochrome P450 CYP6B7 was induced by fenvalerate and involved in fenvalerate detoxification in Helicoverpa armigera. However, the transcriptional regulation of CYP6B7 induced by fenvalerate remains unclear. Here, a series of progressive 5\' deletions of CYP6B7 promoter reporter genes were constructed, and the relative luciferase activities were detected. The results revealed that the relative luciferase activity of plasmid p (-655/-1) was significantly induced by fenvalerate. Further deletion of the region between -655 and -486 bp showed that the highest luciferase activity induced by fenvalerate was observed in plasmid p (-528/-1), while p (-485/-1) had the lowest fenvalerate-induced luciferase activity. Moreover, internal deletion and mutation in the region between -508 and -486 bp resulted in a significant reduction in fenvalerate-induced CYP6B7 promoter activity, suggesting that the cis-acting element responsible for fenvalerate in the CYP6B7 promoter was located between -508 and -486 bp. These results promote an understanding of the expression regulation mechanism of P450 genes that conferring resistance to insecticides.

BACKGROUND: Previous studies indicated that constitutive over-expression of cytochrome P450 CYP6B7 was involved in fenvalerate resistance in Helicoverpa armigera. In this study, the CYP6B7 gene from H. armigera (namely HaCYP6B7), was heterologously expressed in Pichia pastoris GS115. A vector pPICZA-HaCYP6B7 was constructed and transformed into P. pastoris GS115, the transformant of pPICZA-HaCYP6B7-GS115 was then cultured and induced by 1% (v/v) methanol and the heterologous expression of HaCYP6B7 protein in P. pastoris was confirmed by SDS-PAGE and western blot.
RESULTS: Microsomes containing the expressed HaCYP6B7 showed activities against model substrate p-nitroanisole and 7-ethoxycoumarin, with p-nitroanisole O-demethylation (PNOD) and 7-ethoxycoumarin O-deethylation (ECOD) activities of 15.66- and 4.75-fold of the control, respectively. Moreover, it showed degradation activities against the insecticides bifenthrin, fenvalerate and chlorpyrifos, with clearance activities of 6.88-, 1.49- and 2.27-fold of the control, respectively. The interactions of HaCYP6B7 with insecticides were further confirmed by molecular docking in silico with binding scores of 5.450, 5.295 and 2.197 between putative HaCYP6B7 protein and bifenthrin, fenvalerate and chlorpyrifos, respectively.
CONCLUSIONS: The results of present study provided more direct and important evidence on the role of HaCYP6B7 conferring pyrethroid resistance in H. armigera. © 2017 Society of Chemical Industry.