| Conogethes punctiferalis belongs to the Pyralidae family of Lepidoptera,and is a polyphagous insect that feeds not only on herbaceous plants such as corn,soybeans,peanuts,but also on woody plants such as peaches,chestnuts and fruit trees.Peach borer has a strong ability to adapt to the environment and a strong innate immune system,so that when feeding on different hosts in the face of different pathogenic microorganisms without being infected.A variety of antimicrobial peptides(AMPs),which are important immune effectors,can be produced in the body of the larvae.Antimicrobial peptides are the reaction products of innate immunity in biological organisms.As a kind of polypeptides or small molecular proteins with antimicrobial activity produced by the host,antimicrobial peptides can resist the invasion of pathogenic microorganisms and possess broad antibacterial spectrum,unique mechanism of action and good thermal stability.As an immune effector,the expression of antimicrobial peptide genes is often affected by the living environment of the host and greatly increased when stimulated by pathogens.In our previous studies,we found several unknown gene sequences from the transcriptome sequencing results of Conogethes punctiferalis,which were significantly up-regulated in the larvae feeding on different hosts.Considering their bioinformatics characteristics,we speculated they should be antimicrobial peptide genes.In this study,CPAlo-13768 gene,an obvious differential gene in the transcriptome of Conogethes punctiferalis,were choosed to analyse its transcriptional pattern and recombinant protein expression pattern,and the inhibiting effects of both the recombinant protein and synthetic peptides on seven species of fungus infecting corn were determined.The results of the study are expected to provide a basis for host adaptive mechanism of Conogethes punctiferalis and developing insect antimicrobial peptides product.The results are as follows.(1)The coding region of CPAlo-13768 gene was obtained by PCR,and bioinformatics analysis was performed by using related software.The sequence of CPAlo13768 contains a signal peptide sequence,with a theoretical molecular weight of 6.37 kDa and an isoelectric point of 8.29.The protein encoded by CPAlo-13768 gene is a stable hydrophobic protein.The secondary structure of CPAlo-13768 protein consists of three(3-turns and three β-folded structure formed from intramolecular disulfide bonds formed by six conserved cysteine residues.In the sequence alignment of NCBI,no nucleotide sequence homologous to CPAlo-13768 was found,so it was presumed to be a new gene,the GenBank accession number of this gene is MW133083.Amino acid sequence alignment and phylogenetic tree analysis indicated that the protein was similar to Alo series of antimicrobial peptides from Acrocinus longimanus,suggesting that it should have inhibitory effects on fungi.(2)RT-PCR was used to analyze the transcription pattern of CPAlo-13768 gene,and the distribution and expression of CPAlo-13 768 gene in the Conogethespunctiferalis were determined.The expression levels of CPAlo-13768 gene in the adults and the fourth instar larvae were higher than other development stages.In adults,the gene is highly expressed mainly in the head of male and female adults,and in the fat body of larvae.(3)The prokaryotic expression vector pET-32a-CPAlo-13768 was constructed and the expression conditions were optimized.Western blot and WB detection showed that the recombinant protein was effectively expressed in Escherichia coli.The optimal conditions for exogenous expression were obtained as follows:when the OD600 of the broth was at 0.6,added IPTG with a final concentration of 0.5 mM to the broth and induced at 30℃ for 6 h,and the expression quantity of recombinant protein was 108.73μg/mL.After digested and purified by enterokinase,the protein CPAlo-13768 obtained was 11.51 μg/mL,which accounts for about 10.59%of the total recombinant proteins.(4)The inhibitory activity of the antimicrobial peptides recombinant proteins and synthetic peptides against seven kinds of pathogenic fungi infecting maize were identified by mycelial growth rate method.The inhibition rates of antibacterial peptide recombinant protein to fungal pathogens,Fusarium gminearum,Fusarium moniliforme,Bipolaris maydis,Helminthosporium carbonum,Curvularia lunata,Fusarium verticillioides and Exserohilum rostratum,was 11.12%-25.65%,among which the inhibitory effect on Helminthosporium carbonum and Exserohilum rostratum was the strongest,the inhibition rates were 24.12%and 25.65%;The inhibition rates of synthetic polypeptides against 7 species of fungi ranged from 4.59%to 45.13%,of which 13768-1 and 13768-3 had the best inhibition effect on Helminthosporium carbonum,the inhibition rates were 43.44%and 45.13%,13768-2 had better inhibition effect on Curvularia lunata and Helminthosporium carbonum,the inhibition rates were 39.62%and 38.55%. |
