Function Of Cytochrome Monooxygenase Gene AccCYP6k1 In Metabolic Detoxification Of Apis Cerana Cerana

As an important pollinator in nature,Apis cerana cerana or Chinese bees,play an important role in maintaining global biodiversity and providing pollination services for crops.Bee populations around the world have declined dramatically in recent years,and the accumulation of toxic substances(pesticides,metal ions)is one of the main causes of this phenomenon.Therefore,it is of great significance to screen the key genes involved in the metabolic detoxification process of A.cerana cerana and analyze the mechanism of these genes for breeding excellent new varieties of A.cerana cerana,improving their resistance to pesticides and protecting the healthy development of honeybees.Insect cytochrome P450monooxygenase(P450s or CYPs)plays an important role in the metabolic detoxification of endogenous and exogenous substrates,but the mechanism of P450 s in bees is unclear.In this study,a cytochrome monooxygenase gene AccCYP6k1 was isolated from A.cerana cerana.On this basis,the biological function of AccCYP6k1 was systematically studied by biochemical and molecular biology techniques,and the mechanism of AccCYP6K1 gene in metabolism and detoxification of A.cerana cerana was analyzed.The main research results are as follows:(1)Bioinformatics analysis showed that AccCYP6k1 gene was closely related to Am CYP6k1 gene in Apis mellifera and Ad CYP6k1-like gene in Apis dorsata,respectively.The AccCYP6k1 protein sequence contains domains and active sites specific to the insect cytochrome P450 family,such as the C-helix motif,the K-helix motif,and the heme-binding motif,indicating their evolutionary conservation and functional specificity.(2)The spatial and temporal expression pattern of AccCYP6k1 gene was analyzed by reverse transcription quantitative PCR(RT-qPCR).The results showed that AccCYP6k1 gene was mainly expressed in the leg,midgut,head and epidermis of A.cerana cerana,and was expressed in the larval and adult stages,especially in adult workers during the development process,which may be related to the environmental conditions of A.cerana cerana at different days of age.Adult bees,especially workers going out for foraging,are more likely to be exposed to pesticides and other harmful substances.(3)The analysis of the expression characteristics of AccCYP6k1 gene under different pesticide treatments showed that the AccCYP6k1 gene was induced to different degrees under the treatment of beta-cypermethrin,abamectin and imidacloprid,while the expression of AccCYP6k1 was inhibited by three pesticides: propanoate,paraquat and glyphosate isopropylamine.(4)We knocked down the AccCYP6k1 gene in A.cerana cerana through RNAi technology.The results showed that silencing AccCYP6k1 gene significantly increased the susceptibility of A.cerana cerana to abamectin and imidacloprid,and the mortality rate was significantly increased under the treatment of these two pesticides.However,the sensitivity of A.cerana cerana to glyphosate isopropylamine has not changed significantly,which may be related to the specificity of the AccCYP6k1 gene to substrates.(5)The bacteriostatic experiment results showed that the bacteriostatic region on the medium of Escherichia coli expressing recombinant AccCYP6k1 plasmid was significantly smaller compared with the control group.This indicates that overexpression of recombinant AccCYP6k1 protein in E.coli could increase the ability of E.coli to resist environmental stress.(6)After silencing AccCYP6k1 gene,the expression of genes involved in detoxification and oxidative metabolism in A.cerana cerana was significantly down-regulated,and the activities of key detoxifying enzymes and antioxidant enzymes in A.cerana cerana were also significantly decreased.These results suggest that the silencing of AccCYP6k1 gene not only affects the detoxification system of A.cerana cerana,but also may affect the antioxidant capacity of A.cerana cerana,resulting in greater sensitivity to external stress.In summary,this study shows that AccCYP6k1 gene plays an important role in the metabolism and detoxification of A.cerana cerana.Silencing AccCYP6k1 affects the detoxification system of A.cerana cerana,causing accumulation of oxidative damage in the body and increasing the sensitivity to pesticides.The up-regulated expression of AccCYP6k1 gene is related to pesticide resistance,and overexpression of AccCYP6k1 in vitro can increase the host’s tolerance to environmental stress.