Cloning,Prokaryotic Expression And Expression Characteristics Analysis Of Mfe2 From Monochamus Alternatus

This study aims to clone and identify the Mfe2 gene from the Japanese sawyer beetle,Monochamus alternatus,then to express its protein in prokaryotic expression system and to explore its expression patterns in different developmental stages and adult tissues.1.The recombinant plasmid was constructed to clone the Mfe2 gene of M.alternatus using the fast cloning method through designing primers with overlapping area between the vector and target fragment.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 protein mass of 77.56kD.2.The cloned sequence was analyzed with various bioinformatics tools.Bioinformatic analysis showed that MaMfe2 has an AICARFT IMPCHas domain,and shows 91%amino acid sequence identity with peroxisomal multifunctional enzyme type 2 of Anoplophora glabripennis.The expressed Mfe2 protein in prokaryotic expression system was inducted with IPTG and the tissue protein was verified by Western blotting.3.The expression profiles of this gene in different developmental stages and adult tissues were also investigated using the RT-qPCR technology.Developmental expression profiles showed that the expression level of MaMfe2 was the highest in pupal stage,and lower in egg and larval stage,and tissue expression profiles revealed that the expression level of MaMfe2 was the highest in the fat body and lowest in the protothorax of adults.In summary,MaMfe2 encodes 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.