Functional Analysis Of Apis Cerana Cerana Genes AccAMPKα And AccAMPKβ Under Pesticide And Heavy Mental Stress

As a unique bee germplasm resource in China,Apis cerana cerana not only provide abundant bee products,but also plays an important role in plant pollination and ecosystem stability.However,with the development of society and economy in recent years,the pollution of pesticides and heavy metals caused by human activities has posed a serious threat to the development and whole colony health of Apis cerana cerana.Therefore,researching the molecular mechanism of resistance to pesticide and heavy metal stress has great significance for cultivating new varieties of Apis cerana cerana and improving its survival ability under adverse conditions.AMP-activated protein kinase（AMPK）,as an important energy sensor,not only plays an important role in metabolic regulation of biological macromolecules,but also participates in oxidative stress response and is important in the organism’s resistance to external stress.In this study,AccAMPKα and AccAMPKβ genes were isolated from Apis cerana cerana,and their bioinformatics,expression characteristics and functional analysis were analyzed to elucidate the molecular mechanisms in response to pesticide and heavy metal stress.The main results are as follows:（1）AccAMPKα and AccAMPKβ were isolated from Apis cerana cerana.Bioinformatics analysis showed that AccAMPKα and AccAMPKβ were more closely related to Am AMPKαand Am AMPKβ in Apis mellifera.The N-terminus of AccAMPKα contains the conserved kinase domain（KD）and the autoinhibitory domain（AID）,and the C-terminus contained β-binding domain.AccAMPKβ has the conserved glycogen binding domain（GBD）.Genome structure analysis showed that AccAMPKα contained nine exons and eight introns,and AccAMPKβ contained four exons and three introns.The interaction between AccAMPKα and AccAMPKβ was proved by yeast two-hybrid.（2）The expression characteristics of AccAMPKα and AccAMPKβ genes in different tissues and at various developmental stages of Apis cerana cerana were studied.The results of quantitative real-time PCR（q RT-PCR）showed that the expression patterns of AccAMPKα and AccAMPKβ were basically consistent,and the highest expression levels were found in muscle and l day post-emergence adults.（3）The transcript and protein levels of AccAMPKα and AccAMPKβ under different pesticide and heavy metal treatments were analyzed.q RT-PCR results showed that the expression levels of AccAMPKα and AccAMPKβ were affected by different stress conditions.Western blot results showed that abamectin,glyphosate,pyraclostrobine and cadmium chloride treatments induced the expressions of AccAMPKα and AccAMPKβ proteins.The experimental results indicated that AccAMPKα and AccAMPKβ were involved in the response to pesticide and heavy metal stress of Apis cerana cerana.（4）AccAMPKα and AccAMPKβ were silenced in Apis cerana cerana using RNA interference assay,and the survival situation of Apis cerana cerana under pesticide and heavy metal stress was observed and recorded.The experimental results showed that silencing AccAMPKα and AccAMPKβ increased the mortality of Apis cerana cerana under pesticide and heavy metal stress conditions.The results indicated that AccAMPKα and AccAMPKβ play important roles in the resistance to pesticide and heavy metal stress of Apis cerana cerana.（5）AccAMPKα and AccAMPKβ proteins were overexpressed in Escherichia coli（E.coli）and Saccharomyces cerevisiae（S.cerevisiae）.The results showed that compared with the control groups,the number of colonies of E.coli and S.cerevisiae transformed into recombinant plasmid were more in solid medium containing glyphosate,cadmium chloride and hydrogen peroxide.The growth rate of recombinant E.coli in liquid stress medium was significantly higher than the control group.The bacteriostasis test results showed that the inhibition zones of AccAMPKα and AccAMPKβ proteins overexpressed in E.coli were smaller than that of the control group after the cumene hydroperoxide treatment.These results indicated that overexpression of AccAMPKα and AccAMPKβ proteins can improve the endurance of E.coli and S.cerevisiae under different stress conditions（6）After AccAMPKα and AccAMPKβ silencing,the expression levels of some antioxidant genes were decreased,the enzyme activities of peroxidase,catalase,superoxide dismutase and the content of vitamin C were also decreased,while the contents of hydrogen peroxide,malondialdehyde and protein carbonyl were increased.These results indicated that AccAMPKαand AccAMPKβ may respond to stress by participating in antioxidant signaling pathway of Apis cerana cerana.