Cloning And Heterologous Co-expression Of Cytochrome P450s Related Genes From Helicoverpa Armigera And Effects Of Various Compounds On The Binding Of GABA Receptor

Studies showed that insensitivity of target sites, enhancement of enzymes detoxification ability and changes of behavior and morphology are the major metabolisms of insect resistance. As one important detoxification enzyme system, cytochrome P450s can degradate both endogenous and exogenous compounds in organisms, and responsible for the resistance of insect pest to insecticide; Meanwhile, the GABA receptor is one of the primary targets of pesticides, and has been of concern to many researchers. The present study mainly focus on the cloning and heterologous co-expression of cytochrome P450s related genes from Helicoverpa armigera, and the effects of various compounds on the binding of GABA receptor.1. Cloning and characterization of Helicoverpa armigera cytochrome b5reductase (CBR) geneThe full-length of HaCBRl and HaCBR2cDNA with GenBank accession numbers:HQ638220and HQ190046, including969bp and939bp of complete open reading frame (ORF), which encoded322and312amino acids respectively, was cloned with the RACE PCR and RT-PCR. The putative structure and function of HaCBR1and HaCBR2were preliminarily analyzed by SMART program. HaCBRl and HaCBR2showed same C terminal but different N terminal.Under optimized qPCR reaction conditions, EF-la as reference gene to gene transcription level CPR quantitative analysis. The expression of HaCBR1and HaCBR2mRNA was different in most developmental stages of H. armigera except eggs, as well as in tissues such as cuticle, fatbody and midgut. The expression level of the two genes was significantly induced by phenobarbital (PB).2. Cloning and characterization of H. armigera cytochrome P450reductase(CPR) geneThe full-length CPR cDNA is3676bp, containing169bp5’ untranslated region (UTR),2064bp open reading frame (ORF) and1443bp3’ UTR. The ORF of H. armigera CPR was submitted to the GenBank No. HM347785.1. Eighteen different nucleotides were found between HDFR and HDS strain CPR. The typical anchor sructure, which could help the CPR attach the endoplasmic reticulum and maintain the proper spatial structure was predicted; Five conserved domains and three functional domains were found in the third structural model. The H. armigera, beet armyworm (Spodoptera exigua) and cabbage armyworm (Mamestra brassicae) had the highest evolutionary relationship.CPR mRNA was largerly expressed in the HDFR strain intestine; which was2.02times higher in strains of HDS strain. CPR mRNA expression levels in the intestine are significantly different at different developmental stages with highest expression in fifth instar larvae, which is about1.84times the pupal stage, that may be related to physiological bollworm at different developmental stages. CPR mRNA expression levels in intestinal was induced up to17.45times after the HDS strains H. armigera being treated with4mg/g phenobarbital48h. 3. Construction of vector M-pPICZA-CPR-CYP6B7and heterologous co-expression of CYP6B7and CPR in P. pastorisExpression vector M-pPICZA-CPR-CYP6B7was successfully constructed and trasferred into P. pastoris GS115. Recommbinant yeasts with multiple inserts were screened by antibiotic Zeocin and histidine-deficient medium (MD and MM). The results of SDS-PAGE electrophoresis, western blotting, etc, domenstrated that both CYP6B7and CPR were successfully expressed in P. pastoris after1%methanol induction.4. Effects of different compounds on the binding of GABA receptorThe effects of isoxazolines derivatives and other compounds were detected on the binding of [3H]fluralaner,[3H]EBOB and [3H]AVE with the GABA receptor of house fly, honey bee and rat, and the recommbinant of human β3subunit. The results showed that the fluralaner and avermectin could strongly effect the [3H]fluralaner binding on house fly GABA receptor with IC50values0.4and3nM, respectively. The IC50of fluralaner, endosulfan and fipronil to [3H]EBOB on house fly GABA receptor was between39and41nM. Fluralaner and two other isoxazoline derivatives were the best inhibitor to the radioligands binding on GABA receptor, while the other derivatives were poor inhibitors. The IC50of fluralaner, fipronil and heptachlor on rat membrane were>10,000nM,200nM and800nM, respectively, and on the recommbinant of human GABAA receptor β3subunits were not detected,5.4and3.4nM, respectively.The IC50of TETS to [14C]TETS and [3H]EBOB on rat membrane protein binding were0.08μM and0.79μM, respectively. Avermectin, allopregnanolon. isoguvacine, propofol and pyridoxine can inhibit [14C]TETS and [3H]EBOB binding on the rat membrane; however, bicuculline could stimulate the bindings up to59-68%. The correlation of tetramine analogues, pesticides and convulsants between [14C]TETS and [3H]EBOB is0.71.