Expression Characteristics And Functional Analysis Of Ubiquitin-conjugating Enzyme Genes AccUBE2N And AccUBE2G1 In Apis Cerana Cerana

Apis cerana cerana is an excellent bee species unique to China which plays an important role in promoting economic development and maintaining ecological balance.As Apis cerana cerana is sensitive to external stimuli,they are prone to effect by environmental stresses,which is not conducive to the survival and development of the population.Therefore,the research on the resistance and defense mechanism of Apis cerana cerana is of great significance for the sustainable and healthy development of bee population.Ubiquitin-conjugating enzyme E2?UBE2?plays an important role in oxidative stress response,DNA damage repair and regulation of immune response.In this study,AccUBE2N and AccUBE2G1 genes were isolated from Apis cerana cerana and their functions were studied.The relationship between the stress resistance of Apis cerana cerana and the ubiquitin-conjugating enzyme E2 was described at the molecular level.The main results are as follows:?1?Sequence analysis showed that the length of the open reading frame of AccUBE2N is456 bp,encoding a 151-amino-acid protein with a predicted theoretical isoelectric point of5.72 and a molecular mass of 17.18 kDa.The open reading frame length of AccUBE2G1 is507 bp,encoding a 168-amino-acid protein with a predicted theoretical isoelectric point of4.99 and a molecular mass of 19.28 kDa.?2?The amino acid multiple alignment revealed that the UBE2N and UBE2G1 genes have high homology in different insects,and their active cysteine sites are highly conserved.Phylogenetic analysis suggested that both the AccUBE2N and AccUBE2G1 have a close evolutionary relationship with Hymenoptera.We predicted their tertiary structure and found both AccUBE2N and AccUBE2G1 contain UBC domain and belong to class I of E2 family.In addition,AccUBE2G1 contains an acidic ring structure.?3?The transcription factor binding sites related to oxidative stress were found in the promoter sequence of AccUBE2N,indicating that AccUBE2N may play an important role in regulating antioxidant responses;Binding sites of transcription factors related to tissue development and cell stress were found in promoter reogin of AccUBE2G1,suggesting that AccUBE2G1 might participate in tissue specific development and stress protection.?4?qRT-PCR was used to detect the expression characteristics of these two genes in different tissues.The results showed that AccUBE2N and AccUBE2G1 were expressed in different tissues,but the highest expression was in muscles.?5?qRT-PCR was used to detect the expression profiles of AccUBE2N and AccUBE2G1 under various stress factors,the results showed that AccUBE2N and AccUBE2G respond to4?,44?,UV,H₂O₂,heavy metals and pesticides in different degrees.The above results indicated that AccUBE2N and AccUBE2G1 could respond to the cellular stresses caused by different conditions.?6?Western blot analysis was performed to further study the expression patterns of AccUBE2N and AccUBE2G1 under different oxidative stress conditions.The results showed that AccUBE2N and AccUBE2G1 were up-regulated which was consistent with the changes of mRNA level.These preliminary results suggest that AccUBE2N and AccUBE2G1 may play an important role in oxidative stress response.?7?Analysis of the function of AccUBE2N and AccUBE2G1 in vitro by Bacteriostasis assay assay,the results showed that recombinant AccUBE2N protein could enhance the resistance of E.coli cells to heavy metals or peroxides.Recombinant AccUBE2G1 protein has a certain inhibitory effect on the growth of bacteria.