The Function Of Apis Cerana Cerana MKK6-Mediated MAPK Cascades In The Oxidative Stress Response

Mitogen-activated protein kinase（MAPK）signaling pathway is an important signal transduction pathway in the cell,which can be activated by stimuli including cytokines,hormones and various cell stressors,and then regulating cell activities in response to external stimuli.MAPK signaling pathway plays an important role in growth and development,oxidative stress and immune response.Apis cerana cerana is the major honeybee species in China and plays an important role in maintaining the balance of ecological balance and agricultural development.However,the deterioration of natural ecological environment threatens the survival and reproduction of Apis cerana cerana.There,it is of great significance to understand the defense mechanism of Apis cerana cerana in oxidative stress for the development of their population.In this study,we studied the function of MKK6 gene in Apis cerana cerana,and explored the function of AccMKK6 mediated MAPK cascades regulate the oxidative stress response.The results are as follows:The open reading frame of AccMKK6 was isolated from Apis cerana cerana by PCR.The length of the open reading frame is 1002 bp,encoding a polypeptide of 333 amino acid residues.The predicted molecular weight of the protein was about 37.63 kDa,and the theoretical isoelectric point was 7.15.Amino acid sequence alignment and phylogenetic tree analysis showed that the amino acid sequence of AccMKK6 had high homology with other species,and had highly conserved phosphorylation sites and active domains.The expression patterns of AccMKK6 gene were studied by qRT-PCR in various tissues and at different developmental stages of Apis cerana cerana.The results showed that the thorax has the highest expression of AccMKK6 gene,followed by the epidermis and head;AccMKK6 gene was expressed in all developmental stages of Apis cerana cerana,and the expression was higher in pupae stage.AccMKK6 responds to oxidative stress induced by abiotic stress.The expression patterns of AccMKK6 under abiotic stress were investigated by qRT-PCR.The results showed that the transcription level of AccMKK6 gene was induced by abiotic stress treatments such as H₂O₂,heavy metals and pesticides.Western blot results further demonstrated that AccMKK6 was regulated by abiotic stress at the protein level.The E.coli with overexpressing AccMKK6 protein showed increase resistance to abiotic stress.The function of recombinant AccMKK6 protein was analyzed by bacteriostasis experiment.The results showed that the death zones of E.coli with overexpressing AccMKK6 was smaller than that of control group after treatment with H₂O₂ and HgCl₂.The expression of antioxidant genes and the activity of antioxidant enzymes were decreased after the silencing of AccMKK6 gene.When dsAccMKK6 was injected into adult bees,qRT-PCR and Western blot results showed that the expression of AccMKK6 in bees decreased significantly.By detecting the expression levels of AccSOD1,AccSOD2,AccGSTS4,AccCYP4G11,AccGSTO2 and AccGSTD genes,it was found that the transcription of these antioxidant-related genes was downregulated when AccMKK6 was silenced.After AccMKK6 silencing,the activity of SOD and POD in Apis cerana cerana was decreased,but the content of MDA was increased.AccMKK6-Accp38b-Acckayak cascade pathway is involved in the antioxidant stress process of Apis cerana cerana.The downstream gene Accp38b of AccMKK6 was identified by yeast two-hybrid and GST pull-down experiments,and Accp38b was found to interact with Acckayak.It was found that the transcription level of Acckayak gene was induced by abiotic stresses such as H₂O₂,heavy metals and pesticides.At the same time,the transcription levels of Accp38b and Acckayak also decreased after AccMKK6 silencing.