Identification And Function Analysis Of Lebocin-like Gene From The Chinese Oak Silkworm, Antheraea Pernyi

The Antheraea pernyi is a species of wild silkworm insect with important economic value and have been commercially cultivated in China,India,and Korea.It is an irreplaceable important biological resource.Compared to Bombyx mori,due to its particularity of population and growth environment,A.pernyi is highly affected by microbial pathogens.Therefore,studing their antimicrobial immune responses as well as protecting and developing its genetic resources is of great significance to the substainable development of the sericulture industry.Insect antimicrobial peptides(AMPs)play important roles in the innate immune response.In this study,A.pernyi was used as the research object,we initially explored the expression patterns of Ap-lebocin-like gene and its function in innate immunity.Main research contents are as follows:We successfully cloned and identified the Ap-lebocin-like ORF sequence with 489 bp in length.This gene encodes a protein of 162 amino acid residues and should belong to a family of lepidoptera-specific proline-rich antimicrobial peptides.The predicted protein contained many function sites,Blast analysis showed that the lebocin-like protein from A.pernyi has the most similarity with that from Samia ricini(92%identity).The recombinant plasmids pET-28a(+)-Ap-lebocin-like was constructed and transformed to BL21.Soluble recombinant protein was successfully obtained by different concentrations of IPTG.After extensive induction,purification of recombinant protien was conducted by Ni-NTA.Real-time PCR analysis found that Ap-lebocin-like expressed in all the tested tissues,with the highest in the midgut,followed by the epidermis,and the lowest expression was found in the silk gland.Different transcription patterns of the Ap-lebocin-like gene were shown in fat body and midgut after injection of Escherichia coli,AP-NPV,Micrococcus luteus,and Beauveria bassiana.Although Ap-lebocin-like expression is induced by all four microbial pathogens,the maximum mRNA level attained in response to each individual pathogen appeared at different time points.Challenge with M.luteus and B.bassiana induced higher expression of Ap-lebocin-like in both of tissues,compared to challenge with E.coli or AP-NPV Antibacterial activity assay indicated that the Ap-lebocin-like had a high antibacterial activity in vitro,and the antibacterial effect of Ap-lebocin-like on S.aureus(Gram-positive bacteria)is better than that on E.coli(Gram-negative bacteria).These results suggest that Ap-lebocin-like participates in immune response of A.pernyi.