Characterization And Functional Study Of A Cecropin-like Peptide From The Chinese Oak Silkworm Antheraeapernyi

Silkworm is an important silk insect.In present study,a new antibacterial peptide Cecropin-like(ApCec)was screened from the transcriptome of Antheraea pernyi induced by Escherichia coli.The ORF frame of ApCec containing 195 nuclear acids was predicted using software Edit Seq.Tissues of Antheraea pernyi larvae induced by Escherichia coli were collected.The total RNA was extracted and cDNA template was got by reverse transcription.A nucleic acid sequence was gained via amplification,ligation,transformation and sequencing.The alignment showed antimicrobial peptide ApCec gene agrees with the nucleic acid sequence in transcriptome and the gene exists in the body of Antheraea pernyi actually.The full-length ApCec cDNA encoded a protein with 64 amino acids including a putative 22-amino acid signal peptide,a 4-amino acid propeptide and a38-amino acid mature peptide.Amino acid sequence comparison showed that the amino acid sequences between mature antibacterial peptide ApCec and other mature antimicrobial peptide Cecropins were conservative.The publicly reported antibacterial peptide Cecropin amino acid sequences and the predicted antibacterial peptide ApCec amino acid sequence were used to constructe phylogenetic tree.The result revealed that it was clustered into two clusters of Diptera and Lepidoptera.The antibacterial peptide ApCec belongs to the cluster of Lepidoptera and has high homology with other antibacterial peptide Cecropins in smaller cluster.After A.pernyi induced by E.coli for 24 h,tissues of fat body,Malpighian tube,silk gland,midgut,blood cells and epidermis were collected and the distribution of antimicrobial peptides ApCec detected in each tissue by quantitative PCR.The results showed the expression level of antibacterial peptide ApCec gene in fat body,Malpighian tube,blood cells and skin was high,and highest in Malpighian tube.In this study,two peptides pro-ApCec(including the propeptide and mature peptide)and M-ApCec(mature peptide)were synthesized chemically.The purity of peptides were tested by high performance liquid chromatography(HPLC)and molecular weight by mass chemical synthesis,respectively.The antibacterial activity of M-ApCec is more potent than pro-ApCec against E.coli K12 or B.subtilus by MIC and inhibition zone assays.At the same time,the experiment of inhibition zone showed that antibacterial peptide M-ApCec has dose effect against bacteria.Antibacterial peptide M-ApCec was used to treat mouse blood cells which were selected as the representatives of mammalian cells and it was showed that mature antibacterial peptide M-ApCec had low toxicity to blood cells.The structure of antimicrobial peptide M-ApCec were detected by circular dichroism(CD)spectra in hydrophilic or hydrophobic environments and antibacterial peptide M-ApCec displayed to be irregular state in hydrophilic environment and to be ?-helix in hydrophobic environment with the combination of graph and data.Gram negative bacteria E.coli K12 and Gram positive bacteria B.subtilis were dealt with antibacterial peptide M-ApCec and observed by transmission electron microscopy(TEM).The experimental results showed that E.coli K12 and B.subtilis cell membranes were damaged,which indicated that the antibacterial peptide M-ApCec killed bacteria through destroying the integrity of the bacterial cell membranes.The study results show that the antibacterial peptide ApCec plays an important role in A.pernyi to resist the invasion of pathogens,at the same time,through the study of new antibacterial peptide ApCec,it has laid the foundation for for the subsequent more in-depth research and provide a reference to the application for antibacterial drug development and disease control.