Cloning And Functional Identification Of Moricins From The Diamondback Moth,Plutella Xylostella (L.)

In response to infection, insects produce a battery of antimicrobial peptides (AMPs) and other effector molecules to kill the invading pathogens. AMPs are universal in the innate immune systems of both invertebrates and vertebrates, most of these are small cationic molecules consisting of 12-50 amino acids which have broad-spectrum antibacterial property and play crucial roles in the innate immune response. The study of the structure and potential function of AMPs of Plutella xylostella, a notorious pest of cruciferous crops, may provide a theoretical basis for application study of AMPs of P. xylostella and new approaches to pest biologically control. In the present study, we cloned and expressed the moricins from P. xylostella using a TRX (thioredoxin)-6xHis expression system in Escherichia coli. The antimicrobial activity, molecular structure and the possible relationships between the structure and function of moricins were also studied. The main results are as follows:Three full-length cDNAs encoding AMPs of moricins (designated as PxMorl, PxMor2 and PxMorJ) were cloned from P. xylostella, the sequence analysis showed that both of PxMors with an open reading frame (ORF) of 198 bp, which encodes a protein of 65 amino acid residues and have a mature peptides of 42 residues. More interestingly, the mature protein of PxMorl and PxMor2 share the same amino acid residues. Two moricins named PxMor2 and PxMor3 were heterologous expressed and purified. Secondary stucture analysis by Circular dichroism indicated that the two peptides adopt a random coil structure in aqueous solution, but a-helical structure in membrane-like environment. Antimicrobial assays demonstrated that PxMor2 showed higher activity against both gram-negative bacterial E. coli and gram-positive bacterial Staphylococcus aureus, however, PxMor3 only show higher activity against gram-negative bacterial E. coli. Furthermore, we also identified that the mutation of amino acid residues in the N-terminal segment of the ct-helix of PxMors can lead to the change of antimicrobial activity. Transmission election microscopy and confocal laser-scanning microscopy analyses suggest that PxMors can lead to the disruption of bacterial membrane, which might be the reason of the inhibition of bacteria growth.Our results elucidated the antimicrobial activity of moricin in P. xylostella, and the possible relationships between the structure and function of PxMors. The study will benefit for the understanding of P. xylostella AMPs and the immune response, and even provide a new idea for the molecular design of antimicrobial peptides.