Cytochrome P450s Induced By Allelochemicals, Gene Cloning And Expression Of CYP6B6 In Helicoverpa Armigera (H(?)bner)

The content and activity of cytochrome P450s, the total mRNA, expression of CYP6B6 mRNA induced by 2-tridecanone, quercetin and tannic acid, molecular cloning and expression in Escherichia coli of CYP6B6 gene were studied in cotton bollworm, Helicoverpa armigera (Hiibner).1. The induction of the cytochrome P450s by 2-tridecanone and quercetin was investigated in sixth-instar larvae. Combinations of 2-tridecanone and quercetin showed an additive effect on the content of P450s in the midgut, but either an additive or antagonistic effect on the content of P450s in the fat body.2. An 875μl of reaction system, determining the activity of p-nitroanisole O-demethylation by cytochrome P450s, was developed based on the traits of the capillary gas chromatography (CGC) with higher sensitivity, less sample volume and higher resoling power than spectrophotometry. The total reaction system was 875 μl containing 0.1 mol/1, pH 7.8 Tris-HCl buffer, 0.5 μmol NADPH, 7.5 μmol MgCl2, 0.085% BSA, 0.157 mmol p-nitroanisole and 2.08 mg/ml enzyme solution. The p-nitrophenol, product ofp-nitroanisole O-demethylation by cytochrome P450 of the midgut and fat body in the cotton bollwarm larva, was detected by CGC.3. The O-demethylase activity was higher in the fat body than that in the midgut of the sixth-instar larvae fed on the artificial diets mixed with 2-tridecanone and quercetin. The similar results were obtained when induced by the combination of 2-tridecanone and quercetin at different concentrations. When the concentration of inducers, mixture of 2-tridecanone and quercetin, was 0.5 mg/ml in the diet, the maximum 0-demethyiase activities of P450s in the midgut and fat body were 2.6 and 3.88 times higher than those in the relevant control, respectively. There was no synergism between the effects of two allelochemicals, which both increased significantly the activities of P450s in midgut and fat body.4. The time-course and dosage-course of P450s O-demethylase activities were determined in the larvae treated by quercetin and 2-tridecanone. P450s activities induced by low dosage of quercetin, in midgut and fat body, dropped firstly and then increased. Up to 48 h, activity reached the maximum, which 2.03 and 2.4 times higher than those in the relevant control, respectively. Under the high dosage, activities at 48 h reached the maximum, which 2.8 and 2.79 times higher than those in the relevant control, respectively. P450s O-demethylase activities in fat body of the larvare induced by the high dosage of 2-tridecanone were higher than those by low dosage from 4 h to 48 h, 2-tridecanone obviously inhibited P450s O-demethylase activities at 60 h and 72 h. 2-tridecanone had no effect on P450s activities of midgut compared with the relevant control, except that P450s activities by 0.1 mg/ml 2-tridecanone increased significantly at 4 h and 36 h.5. The effect of quercetin on the total mRNA was investigated, the results showed that the total mRNA was obviously higher than the corresponding control at 5 h, 24 h and 36 h in the midgut, and it had no significant changes in the fat body compared with the control.6. A semi-quantitative RT-PCR method was used to determine the expression level of CYP6B6 mRNA in the midgut and fat body of the larvare induced by by 2-tridecanone, quercetin and tannic acid. The expression levels of CYP6B6 mRNA were obviously correlated with the concentrations of 2-tridecanone, but no correlation with the concentrations of quercetin. No induction of CYP6B6 mRNA expression by tannic acid in fat body was observed, but significant induction was found in midgut.7. Using two pairs of primers designed according to sequence of CYP6B6, we cloned full length of P450 from midgut and fat body tissue in cotton bollworm by RT-PCR. According to biological informatics analysis, the P450 genes (GeneBank accession number, AY950636 and AY950637) belong to CYP6B6.8. Expression vector pGEX-4T-I was used to express the CYP6B6 genes from the cotton bollworm in Echerichia coli; SDS-PAGE and CO difference spectrum analysis showed that t...