Isolation And Expression Analysis Of AccCYP336A1 And AccCYP18A1 Genes In Apis Cerana Cerana

Cytochrome P450 monooxygenases(P450), widely distributed multifunctional enzymes, that play an important role in the pesticide metabolism and molting hormone synthesis and closely related to the growth, development and defense of the insect. Studies have found that these enzymes show peroxidase-like activity and may thus be involved in protecting organisms against reactive oxygen species(ROS). In this study, we selected the economically important and our country unique species Apise cerana cerana as the experiment material, and two genes belong to the CYP3 clade and CYP2 clade of cytochrome P450 monooxygenases, AccCYP336A1, AccCYP18A1, have been cloned by RT-PCR and RACE-PCR according to the homologous sequence of Apis mellifera. Then, a series of studies have been conducted on the sequence and expression analysis, function prediction of two genes. The main results are as follows:(1) The sequence analysis indicated that the open reading frame(ORF) of AccCYP336A1 was 1491 base pair(bp). The ORF encoded a polypeptide of 496 amino acids with a predicted molecular mass(Ms) of 57 kDa and a theoretical isoelectric point of 8.8. The ORF of AccCYP18A1 was 1620 bp. The ORF encoded a polypeptide of 539 amino acids with a predicted Ms of 60.7 kDa and a theoretical isoelectric point of 8.42. Alignment analysis revealed that AccCYP336A1 and AccCYP18A1 shared high similarities with other known species. Conserved characteristic regions were identified in their amino acid sequences, respectively. The evolutionary tree analysis found that AccCYP336A1, AccCYP18A1 belong to CYP3 clade and CYP2 clade, respectively.(2) The qRT-PCR was performed to examine the developmental regulation and tissue distribution of AccCYP336A1 and AccCYP18A1. qRT-PCR showed that AccCYP336A1 was expressed in the whole period of metamorphosis and the highest mRNA level of AccCYP336A1 was detected in egg. AccCYP18A1 was expressed in the whole period of metamorphosis and the highest expression level was detected in the differentiation stages of development such as fifth instars larvae and 1-day old adult. These results suggest that AccCYP336A1 and AccCYP18A1 might play an important role in the transitions of metamorphosis development. The results of tissue distribution revealed that AccCYP336A1 expression was highly concentrated in the epidermis and AccCYP18A1 expression was highly concentrated in the midgut. The results preliminary revealed the tissue specificity of these two genes.(3) qRT-PCR showed that the transcript levels of AccCYP336A1 and AccCYP18A1 could be markedly accumulated by treatment with various abiotic stimulis such as high temperature, low temperature, ultraviolet ray and pesticides. The results indicated that AccCYP336A1 and AccCYP18A1 might be involved in responses to abiotic stresses, and play an important role in antioxidant.(4) Western-blot showed that the translational levels of AccCYP336A1 and AccCYP18A1 can be markedly accumulated by treatment with various abiotic stimulis such as low temperature, ultraviolet ray and pesticides. The results were consistent with the features of transcription expression level and verified that AccCYP336A1 and AccCYP18A1 may be involved in antioxidant.(5) Using the disc diffusion assay testing the function of vitro AccCYP336A1 in E.coli. The study found that the Escherichia coli(E.coli) strains contains AccCYP336A1 could improve the resistance of paraquat.