Molecular Cloning, Characterisation And Expression Of Methionine Sulfoxide Reductase A Gene From Apis Cerana Cerana

ROS generated by stimulation lowers the cellular antioxidant capacity, alters protein function and damages proteins. Methionine can be oxidized to methionine sulfoxide, which can lead to reduced biological activity of proteins which or loss. Methionine sulfoxide reductases (Msrs) catalyze the reduction of methionine sulfoxide (MetO) to methionine and play key roles in protein repair and reactive oxygen species (ROS) scavenging. Previous studies of MsrAs have mainly focused on antioxidant defenses and modulation of the aging process, and little information about MsrA in bees has been available until now. Here, a novel MsrA gene designated AccMsrA was isolated from Apis cerana cerana. Quantitative real-time PCR (qRT-PCR) and Immunohistochemical localization were used study the AccMsrA mRNA expression in different developmental stages and tissues. Furthermore, the accumulation of AccMsrA mRNA was induced by oxidative stimulation . The results as follow:1. Based on the conserved region of insect MsrA genes, primers were designed, and then RT-PCR and RACE-PCR were performed to amplify the region of AccMsrA. The full-length cDNA of AccMsrA (GenBank accession no. HQ219724) is 1,540 bp, and it contains a 136-bp 5′untranslated region (UTR) and a 753-bp 3′UTR. The cDNA harbors a 651-bp open reading frame (ORF) encoding a protein of 217 amino acids. The theoretical molecular mass of AccMsrA was calculated as approximately 25.0 kDa. Sequence alignment analysis showed that AccMsrA shares high similarity with other insect MsrAs. A multiple sequence alignment showed that AccMsrA shares 49.6%, 45.8% and 51.3% identity with the MsrAs from Aedes aegypti, Drosophila erecta and Culex quinquefasciatus, respectively. The highly conserved region GCFW, which is the fingerprint of MsrAs, was found in the N-terminus. Moreover, a cysteine residue was identified in the conserved GCFW region that is involved in the catalytic mechanism. 2. Analysis of the 5′flanking region of AccMsrA revealed a group of putative cis-acting elements that are related to the regulation of development and responses to external stimuli. Quantitative real-time PCR (qRT-PCR) detected high levels of AccMsrA mRNA during pupation (fifth-day larvae) and in the head tissue of adults. Immunohistochemical localization showed that AccMsrA was primarily concentrated in the abdomen and intestinal tract wall in fifth-instar larva. Furthermore, the expression of AccMsrA was up-regulated by multiple oxidative stresses, including ultraviolet (UV)-light (30 mj/cm²), heat (43℃) and H₂O₂ (2 mM). These results indicate that AccMsrA might fulfill an important role in the regulation of insect development and in response to various environmental stresses.3. The complete ORF sequences of AccMsrA were cloned into the pET30a (+) expression vector. The recombinant expression vectors were transformed into E. coli strain BL21 (DE3) and then induced. The 29 KDa recombinant proteins were separated and purified. Antisera were produced by injecting purified recombinant AccMsrA protein into white mice.4. The Immunohistochemical localization results revealed that AccMsrA was widely distributed, and high activity was detected in the abdomen and the intestinal tract wall, in fifth-day larvae. This result implies that AccMsrA might be involved in the the growth of segmental ventral organs in pupae.