Isolation And Functional Analysis Of Stress Tolerance-Related Genes AccMKP3 And AccApoD In Apis Cerana Cerana

Apis cerana cerana is a unique local bee species in China with great economic and social benefits.However,their survival is seriously threatened by various external stresses and its population is decreasing.In order to better protect the precious germplasm resources of Apis cerana cerana,it is imperative to study the stress tolerance-related genes in bees by molecular biology.MAP kinase phosphatases(MKPs)and Apolipoprotein D(ApoD)which participate in the response of cells to various stimulis,play an important role in the process of growth and development,immune response,oxidative stress and physiological response related to various diseases.In this study,we isolated the AccMKP3 and AcccApoD genes in Apis cerana cerana and explored their functions.The main results are as follows:(1)The coding region of AccMKP3 and AccApoD were obtained by RT-PCR.The coding sequcnce of AccMKP3 is 1203 bp,encoding a 400-amino-acid protein with a predicted theoretical isoelectric point of 4.90 and a molecular mass of 43.922 kDa.The coding sequence of AccApoD is 714 bp,encoding a 237-amino-acid protein with a predicted protein molecular weight of 27.7 kDa and a predicted isoelectric point of 8.42.(2)The amino acid sequence alignment and phylogenetic tree analysis showed that MKP3 and ApoD were highly conserved across different insects,and AccMKP3 and AccApoD in Apis cerana cerana were most closely related to MKP3 and ApoD in Apis mellifera.Promoter sequence analysis showed that the promoter regions of AccMKP3 and AccApoD contain a variety of transcription factor binding sites involved in growth and stress responses.Genomic structural analysis showed that both AccMKP3 and AccApoD contain 4 exons and 3 introns.(3)The developmental and tissue-specific analysis of AccMKP3 and AccApoD were carried out by qRT-PCR.The results showed that they were expressed in all developmental stages of Apis cerana cerana,AccMKP3 was expressed highest in the white-eyed pupae while AccApoD was expressed highest in the prepupae stage,respectively.Additionaly,these two genes were expressed in many tissues of Apis cerana cerana.AccMKP3 was highly expressed in the thorax of adult bees,while AccApoD was highly expressed in the head and chest of adult bees.(4)The expression patterns of AccMKP3 and AccApoD under different stress conditions were analyzed by qRT-PCR.The results showed that AccMKP3 and AccApoD were significantly induced by temperature,UV,heavy metals and pesticides,indicating that they play important roles in the protection of Apis cerana cerana against environmental stress.(5)The bactericidal tests of recombinant AccMKP3 protein and recombinant AccApoD protein showed that overexpression of these two proteins made Escherichia coli more sensitive to oxidative stress,indicating that these two proteins may have antibacterial activities.(6)Following the silenced of the AccMKP3 and AccApoD gene in Apis cerana cerana by RNAi,respectively,the transcriptional levels of some antioxidant genes were significantly changed,and the activities of antioxidant enzymes such as POD,SOD and CAT were significantly increased,indicating that these two genes have antioxidant effects and play an essential role in maintaining the redox balance of Apis cerana cerana.