Cloning, prokaryotic expression and expression profiling of Mfe2 from the Japanese sawyer beetle, Monochamus alternatus (Coleoptera: Cerambycidae)
LIU Xiao-Long1,2, ZHANG Bin2, TIAN Hao-Kai2, NING Jing2,3, WANG Hai-Xiang1,*, ZHAO Li-Lin2,*
(1. College of Forestry, Shanxi Agricultural University, Taigu, Shanxi 080301, China; 2. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; 3. College of Life Sciences, Hebei University, Baoding, Hebei 071002, China)
Abstract 【Aim】 Mfe2, peroxisomal multifunctional enzyme type 2 gene, plays a very important role in the β-oxidation of fatty acids. This study aims to clone and identify the Mfe2 gene from the Japanese sawyer beetle, Monochamus alternatus, to express its encoded protein in prokaryotic expression system, and to explore its expression patterns in different developmental stages and adult tissues of the beetle.【Methods】 The recombinant plasmid was constructed to clone the Mfe2 gene of M. alternatus using the fast cloning method through designing primers with overlapping region between the vector and target fragment, and the cloned sequence was analyzed with various bioinformatics tools. The expressed Mfe2 protein in prokaryotic expression system was inducted with IPTG, and the fusion protein was verified by Western blotting. The expression profiles of this gene in different developmental stages and adult tissues of M. alternatus were investigated using the RT-qPCR technology. 【Results】 Mfe2 was cloned from M. alternatus and named MaMfe2 (GenBank accession no.: MF944109). Its open reading frame is 2 148 bp, encoding a protein of 716 amino acids, with an estimated molecular mass of 77.56 kD. Bioinformatics analysis showed that MaMfe2 has an AICARFT_IMPCHas domain, with 91% amino acid sequence identity with peroxisomal multifunctional enzyme type 2 of Anoplophora glabripennis. The expressed protein in BL21 (DE3) strains of Escherichia coli bound specifically with its antibody in Western blot analysis. Developmental expression profiles showed that the expression level of MaMfe2 was the highest in the pupal stage, and lower in egg and larval stages, and tissue expression profiles revealed that the expression level of MaMfe2 was the highest in the fat body and the lowest in the protothorax of adults. 【Conclusion】 MaMfe2 encodes a peroxisomal multifunctional enzyme in M. alternatus, and its expression level is various in different developmental stages and adult tissues. This study provides the basis for further research on the function of this gene in M. alternatus.
LIU Xiao-Long,ZHANG Bin,TIAN Hao-Kai et al. Cloning, prokaryotic expression and expression profiling of Mfe2 from the Japanese sawyer beetle, Monochamus alternatus (Coleoptera: Cerambycidae)[J]. ACTA ENTOMOLOGICA SINICA, 2018, 61(3): 282-291.